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CHARACTERIZATION OF THE FECAL INDICATOR 
BACTERIAL FLORA OF SANITARY SEWAGE VVITI 
APPLICATION TO IDENTIFYING THE PRESENCE 01 
SANITARY WASTE IN STORM SEWERS 



R. A. C. PROJECT NO. 247PL 





H 




Environment 
Ontario 



ISBN 0-7729-8769-6 



CHARACTERIZATION OF THE FECAL INDICATOR 
BACTERIAL FLORA OF SANITARY SEWAGE WITH 
APPLICATION TO IDENTIFYING THE PRESENCE OF 

SANITARY WASTE IN STORM SEWERS 



R. A. C. PROJECT NO. 247PL 



Prepared for Environment Ontario by: 

Patricia Seyfried and Trudy Bleier 
Department of Microbiology 
University of Toronto 

Elizabeth Harris 

Lake Simcoe and Region Conversation Authority 

Michael Young 

Ministry of the Environment Laboratory Services 

.SEPTEMBER 1991 

o 

RECYCLABLE 

Cette publication technique n'est disponible qu'en anglais. 

Copvright: Queen's printer for Ontario, 

1991 

This publication may be reproduced for 

non-commercial purposes with appropriate 

attnbution. 



PIBS 1657 

log 91-9300-274 



ACKNOWLEDGEMENT AND DISCLAIMER 



This report was prepared for the Ontario Ministry of the 
Environment. The views and ideas expressed in this report 
are those of the author and do not necessarily reflect 
the views and policies of the Ministry of the 
Environment, nor does mention of trade names or 
commercial products constitute endorsement or 
recommendation for use. The Ministry, however, 
encourages the distribution of information and strongly 
supports technology transfer and diffusion. 

Any person who wishes to republish part or all of this 
report should apply for permission to do so to the 
Research and Technology Branch, Ontario Ministry of the 
Environment, 135 St. Clair Avenue West, Toronto, Ontario, 
M4V 1P5, Canada. 



Copyright: Queen's Printer for Ontario 

This publication may be reproduced for 
non-commercial purposes with appropriate 
attribution. 



ACKNOWIEDGEMENT 

Itds work was supported by research project nuinber 274 PL from the Ontario 
Ministry of the Environment. The itanuscript was prepared by Elizabeth Harris, 
Patricia Seyfried and Trudy Bleier. Technical assistance was provided by 
Bhavna Madhvani, Anjini Prashad, Rita Harmandayan, Joanne Vavougios, Mona 
Sidarous, Nella Mauceri, Julianna Giczi, Kay Auanjou, Lionel Noronha, Seema 
Boodoosin^, Bruno Esposito, Kay Hoasjoe, Aisha SheiWi, Helen Qiao, In-ja Huh, 
Jim Ng, Niva Kulendran, Eric Hani, Natalie Veitch, Eric Bauer, Arif Somani, 
Frank Fassos, Yan Xu, Frank De Palma, Charles Wachsberg and Susan Davison. 

Collection of samples by Mr. Alex Marich and employees of the City of 
Toronto Public Works Department is gratefully acknowledged. 

DISCIAIMER 

This report has been prepared for the research Advisory Committee, 
Ministry of the Environment in fulfillment of the terms of the grant. The 
views ej^ressed herein are those of the authors and they do not necessarily 
reflect the views and policies of the Ontario Ministry of the Environment. 

HJBUCATIONS RELATED TO THIS roCJECT 

Seyfried, P.L, E.M. Harris, I. Huh, R. Harmandayan and E. Hani 1987. 
Characterization of the fecal indicator bacterial flora of sanitary sewage 
with application to identifying the presence of sanitary waste in storm 
sewers. Proceedings Technology Transfer Conference, Part D. Analytical 
Methods. Royal York Hotel, Toronto, November 30, and December 1, 1987. 
Sponsored by the Research Advisory Committee, Ministry of the Environment, 
pp. 1-24. 



- 11 - 



Seyfried, P.L. , T. Bleier, Y. Xu and R. Harmandayan. 1988. C3Tarac±erization 
of the fecal indicator bacterial flora of sanitary sewage with application 
to identifying the presence of sanitary waste in storm sewers. 
Proceedings Technology Transfer Conference, Session B, Water Quality 
Research, Royal York Hotel, Toronto, November 28 and 29, 1988. Sponsored 
by Research and Technology Branch, Environment Ontario, pp. 247-267. 



- Ill - 
ABsrnRAcr 

storm sewers are designed to channel untreated storm water into surface 
waters. An unusually hi<^ dry weather flew of storm sewage along with hi(^ 
fecal coliform counts irjiicate that there may be potentially hazardous sanitary 
connections in the storm sewer line. Ihe c±ijective of this study was to assess 
the use of bacterial indicators to trace illegal sanitary connections to storm 
sewers. The indicators selected for study were Escherichia coli, fecal 
coliforms, fecal streptococci, enterococci, Pseudomonas aeruginosa, Clostridium 
perfrinqens , and Bifidobacterium sp. The organisms were collected during 
periods of wet and dry weather from October, 1986 to August, 1988. The 
sampling sites A, B and C in the Mount Steven Trunk storm sewer line were 
selected because this area was designated a hi(^ priority sewer by the Ministry 
of the Environment. Non-priority sites, labelled X, Y and Z in the Mount 
Steven Trunk storm sewer branch lines were saitpled for conparison. For wet 
weather, storm water run-off was collected at the X, Y and Z sites. Samples 
were also obtained from a sanitary s^^er in close proximity to the priority 
storm sewer sanpling points; these sites were labelled D, E and F. 

Biochemical testing, serotyping, and/ or genotyping were used to further 
characterize more than 4,000 fecal streptococcus, Pseudomonas aeruginosa , and 
Bifidobacterium spp. isolates. Speciation of the fecal streptococci showed 
that Streptococcus faecal is subsp. faecal is was more predominant in sanitary 
and high priority sewers than in surface runoff and non-priority sewers. 
Streptococcus faecium subsp. casseliflavus , on the other hand, was found 
primarily in surface water runoff and non-priority storm sewers. Litmus milk 
reactions among the S^ faecal is isolates generally did not assist in source 
tracing. IXIA sequence studies of the fecal streptococci, using Restriction 



- IV - 

Endonuclease Analysis (REA) , produced many different restriction patterns and 
it was difficult to establish any relationship between the isolates. Although 
serotyping of Pseudomonas aeruginosa was found to produce nonspecific results, 
genotyping did provide a precise method of fingerprinting the organisms. 
Atteirpts to genotype the bifidobacteria were unsuccessful; however, members of 
the genus show promise as pollution indicators because they were found in high 
concentrations in human feces and sanitary sewage. Further work to determine 
the natural sources of Bi f idobacter ium species should be carried out. 



- V - 
COKUJSlOtJS 

The results of the study showed that the levels of bacterial indicators, 
particularly fecal coliforms, Escherichia coli . Pseudomonas aeruginosa . and 
Bi f idobacter ium spp., were hi(^ in the high priority storm sewer line at or 
near saitpling points A, B and Y. The data suggest that there is an impact near 
site A in the storm sewer line that itay be due to human fecal pollution. 

Characterization of the indicator bacteria by means of biochemical 
testing, serotyping and genotyping produced notable results. For exanple, 
speciation of the fecal streptococci was shown to be a useful means of 
identifying sewer or storm sewer water content. The Streptococcus species S^ 
faecium tended to be equally represented in all types of sanples. 
Streptococcus faecal is subsp. faecal is . on the other hand, was found with 
greater frequency in sanitary and priority storm sewers than in surface runoff 
and non-priority sewers. In contrast, S^ faecium subsp. casseliflavus was 
almost nonexistent in sanitary sewage but was the predominant enterococcus in 
non-priority storm sewer water. 

Mundt's 1973 S^ faecal is studies showed that isolates from non-human 
sources produced proteinization reactions in litmus milk whereas the isolates 
from human feces yielded acid curds. Such a trend was not observed among the 
Si faecal is species isolated in this investigation and therefore the litmus 
milk reactions do not appear to be a useful criterion in source tracing. As 
well, genotyping of the faecal streptococcal isolates from the sanitary sewer, 
priority and non-priority storm sewers and the surface nmof f did not produce 
conclusive resiiLts. A total of 64 different restriction patterns were 
identified among the 192 streptococcal isolates examined and the patterns did 
not show any distinctive trends. 



- VI - 

Fecal col i form to fecal streptococcus ratios have been proposed as a 
method of estimating whether the source of pollution was from a human or 
nonhuman source. The FC/FS ratios were calculated during the course of this 
study but the results were inconclusive. Patios greater than 4.0, suggesting a 
human source, were consistently found in the sanitary sewage but the ratios in 
the remainder of the saiiples varied too widely for any assuirptions to be made. 

Pseudomonas aeruginosa counts were highest in sanitary and priority storm 
sewage; they reached their lowest levels in non-priority storm sewage. 
Serotype 0:6 was the predominant type in all samples collected. Also evident 
were serotypes 0:1, 0:10, 0:11, 0:4, 0:3 and 0:2. Ihere was a tendency for 
serotype 0:10 to be found primarily in sanitary sewage, but otherwise 
Pseudomonas serotyping did not appear to be applicable to source 
differentiation. Genotyping P^ aeruginosa organisms, on the other hand, did 
produce a more precise method of fingerprinting the isolates. For example, 
the ei(^t different genotypes that were obtained from the sanitary and hii^ 
priority storm sever cultures were not found in the non-priority sewer and 
surface water runoff isolates. Also, similar REA patterns were observed in the 
P. aeruginosa isolates from storm water runoff and the corresponding storm 
sewer site. 

Studies in our laboratory have shown that Bi f idobacterium spp. are present 
in human feces in concentrations of approximately 1 x 10^ per gram. The 
results of this investigation showed that bifidobacteria counts were in the lO'^ 
to 10^ per lOOmL range in sanitary and hi(^ priority storm sewage and decreased 
by two orders of magnitude in non-priority storm sewage. Mara and Oragui 
(1983) have suggested that sorbital fermenting bifidobacteria (i.e B^ 
adolescentis and B^ breve) could be used as indicators of human fecal 



- Vll - 

pollution. Hcwever, we would suggest caution in this approach because we have 
been able to isolate B^. adolescentis fran dog feces and B. breve, B^ minimum 
and Bi thermo]::hilum from chicken fecal saitples. It is interesting that the 
nonhuman B^ thermophilum strain was also isolated from the non-priority storm 
sewer at site Z. 

Although three different restriction enzymes were used to digest v^ole 
cell CNA from the bifidobacteria not enough CMA was recovered and the 
genotyping experiments were unsuccessful. This aspect of the investigation 
merits further study. 

REOMlEIlEftnCNS 

1. This project shewed that source determination studies can be conducted 
most effectively during periods of dry weather when there is a substantial 
flow in the storm sewer line. During storm events it is difficult to 
trace the source of contamination because the bacterial indicatoirs come 
from a variety of sources. It is therefore recommended that source 
tracing be done only under dry weather conditions. 

2. The results of this investigation suggest that because S^ faecium subsp. 
casseliflavus was isolated primarily frcsn surface water runoff and non- 
priority storm sewers it may come solely from nonhuman sources. This 
concept is supported by the fact that we have been unable to isolate the 
organism from a limited number of human fecal specimens. We propose that 
further studies be carried out to determine the source of this organism. 
Also, that field studies be done to assess the applicability of a E^ coli 
to S^ casseliflavus or a Bifidobacterium to S^ casseliflavus ratio. 

3. It was apparent from this project that the serotyping of P^ aeruginosa 



- Vlll - 

isolates did not aid in the tracing of illegal sanitary connections in 
storm sewers. It is suggested that in future studies serotyping be 
combined with phage typing or genotyping to fingerprint the Pj^.ndnmnn^i?^ 
organisms. A probe, developed from either total chramoscaral CNA or a 
specific fragment common to human strains, might allow us to hybridize 
against all other strains and differentiate between human and nonhuman 
fecal material. « 

Ihe project results suggest that bifidobacteria show promise as pollution 
indicators. Additional work should be done to determine the natural 
habitat of the individual Bifidobacterium species. In addition, further 
attenpts could be made to genotype the organisms. 



Table of Oorttents 



Page No. 



INHOXJCnCN 

Rationale for the Use of Selected Indicator Organisnis 1 

Fecal Coliforms 3 

Escherichia coli 3 

Fecal Streptococci and Enterococci 5 

Fecal Coliform to Fecal Streptococci Ratios 6 

Pseudomonas aeruginosa 8 

Bifidobacterium spp. 8 

Clostridium perfrinqens " 9 

Scope 9 

METQKOS 10 

Sampling Sites 10 

Sarrple Collection 10 

Bacterial Isolation and Enumeration 13 

Fecal coliforms, E. coli 
Fecal streptococci 
Enterococci 
Pseudomonas aeruginosa 
Bi f idobacter ium spp. 
Clostridium perfringens 
BACTERIAL CHARACTERIZATICN 15 

Fecal streptococci; Serotyping of Pseudomonas aeruginosa 
Genotyping 15 

Fecal streptococci 

Ps . aeruginosa 

Bifidobacteria 



Page No. 

RESUIirS AND DISCLJSSICN 18 

1986 and 1987 Sxxrveys 18 

Fecal Indicator Bacteria 

Pseudomonas aeruginosa 

Bifidobacteria 

Clostridium perfringens 

FC/FS Patio 

SUMMARY 36 

Summary of 1986 - 1987 Survey Results 36 

1988 Surveys • 37 

Summary of 1988 Survey Results 47 

DISCUSSICN OF ADDrnCNAL roCOECIS 50 

RKFEKhUCES 55 

APFQIDIX A 60 

Tables 14-69 60 

APFQTOIX B 116 

Corrparative Study of the Survival of Indicator 
Bacterial Species by Eric Bauer 

APFQIDIX C 172 

The Isolation and Identification of Bifidobacteria 
from Fecal and Sewage Samples by In-ja Huh 

APETNDIX D _ 203 

A Study of the Survival of Bifidobacteria and their 
Role in Water Quality Control by Sheila Shibata 



Page No. 

APFQIDIX E 241 

Clostridium perfrincrens and Bi f idobacter ia sp. as 
Tracers in Storm Sewers by Eiric Hani 

APPENDIX F 254 

Characterization of Pseudomonas aeruginosa from Storm 
and Sanitary Sewers by Rita Harmandayan 

APPENDIX G 294 

Bifidobacterium sp. 
APPENDIX H 299 

Fecal Strepjtococci 
APPENDIX I 307 

Pseudomonas aeruginosa 
APPENDIX J 312 

Media Preparation 



UST OF FIGURES 
Figure Page No. 

1 Sairpling sites in Mcunt Steven storm sewer and 12 
sanitary sewer lines 

2 Fecal coliform levels at the storm sewer and 20 
sanitary sewer sairpling sites 

3 Fecal strepotococcus levels at the storm sewer 21 
and sanitary sewer sairpling sites 

4 Pseudononas aeruginosa levels at the stonri sewer 23 
and sanitary sewer saitpling sites 

5 Bifidobacteria levels at the storm sewer and 25 
sanitary sewer sairpling sites 

6 Clostridium perfrinqens levels at the storm sewer 26 
and sanitary sewer sairpling sites 

7 Percentage distribution of Streptococcus faecal is 48 
subsp. faecal is among the sanitary sewer sites 

(D,E,F), the priority storm sewer (A,B,C,Y), the 
non-priority storm sewer (X,Z) and the storm water 
runoff (P,G,R,Q,) locations 

8 Percentage distribution of Streptococcus faecium subsp. 49 
casseliflavus among the sanitary sewage sites (D,E,F) , 

the priority storm sewer (A,B,C,Y), the non-priority 
storm sewer (X,Z) and the storm water runoff (P,G,R,Q) 
locations 

APPENDIX 

B-1 Colony Forming Units per mL vs. Time at Room 139 

Tenperature 

B-2 Colony Forming Units per mL vs. Time at 140 

15 degrees Celsius 

B-3 E. coli CRJ/mL vs. Time at Room Tenperature 141 

and 15 degrees Celsius 

B-4 Pi aeruginosa CKJ/mL vs. Time at Room 142 

Temperature and 15 degrees Celsius 

B-5 Strep , on m-Ent CFU/mL vs. Time at Room 143 

Tenperature and 15 degrees Celsius 

B-6 Strep , on m-E CFU/mL vs. Time at Room 144 

Tenperature and 15 degrees Celsius 

B-7 Bi loncaim CFU/mL vs. Time at Room Tenperature 145 

and 15 degrees Celsius 



APFQJDIX 

Figure Page No. 

B-8 Magnification of Colony Forming Units per mL vs. 146 

Time at Room Teitperature and 15 degrees Celsius 

B-9 Magnification of Colony Forming Units per mL vs. 147 

Time at Room Temperature and 15 degrees Celsius 

B-10 Magnification of Ei coli CFU/mL vs. Time at Room 148 
Teitperature and 15 degrees Celsius 

B-11 Magnification of P^ aeruginosa CFU/mL vs. Time 143 

at Room Tenperature and 15 degrees Celsius 

B-12 Effect of 0.2 ppm Oilorine on Various Organisms 151 

at a pH of 6.0 

B-13 Effect of 0.4 ppm Qilorine on Various Organisms 152 

at a pH of 9.0 

B-14 Effect of 0.2 ppm Chlorine on Various Organisms 153 

at a pH of 9.0 

B-15 Effect of 0.4 ppm Oilorine on Various Organisms 154 

at a pH of 9.0 

C-1 Geometric mean concentration of FC, E^ coli and 188 

Bifidobacteria in storm sewage 

C-2 A comparison of the irecovery of Bifidobacteria 193 

species from feces and sewage on two selective 
media. 

C-3 Percentage species of Bifidobacteria in feces, 195 

sanitary and storm sewage 

D-1 Dialysis membrane diffusion chamhser. 216 

D-2 In vitro survival of bifidobacteria isolated 224 

from sewage and E^ coli in Lake Ontario water. 

D-3 In vitro survival of bifidobacteria isoalted 226 

from human feces and E^ coli in Lake Ontario 
water. 

D-4 In vitro survival of Bifidobacterium bifidum 227 

(ATDC #696) and E^ coli in lake Ontario water 



APFQOIX 

Figure Page No. 

D-5 In vitro survival of Bi f idobacter ium breve 228 

(ATCC #701) and E. coli in Lake Ontario water 

D-6 In vitro survival of fecal bifidobacteria and 229 

E. coli in lake Ontario Water 

E-1 Geometric Mean Concentrations of Fecal 248 

Coli forms, E^ coli, Bifidobacteria spp. and 
Clostridium perfringens in Sanitary Sewage 
during Dry Weather Survey June 10, 11 
and 12, 1987 

E-2 Geometric Mean Concentrations of Fecal 249 

Coli forms, E^ coli . Bifidobacteria spp. and 
Clostridium perfrinqens in Hii^ Priority 
Storm Sewage during Dry Weather Survey 
June 10, 11 and 12, 1987 

E-3 A coitparison of the Geometric Mean 250 

Concentrations of E^ coli , Clostridium 
perfrinqens and Bifidobacteria in High 
Priority and Non-Priority Storm and 
Sanitary Sewage (IDG values) 

E-4 A conparison of the Geometric Mean 251 

Concentrations of E^, coli , Clostridium 
perfrinqens and Bifidobacteria in Human, 
Cat and Dog Feces (HDG values) 

E-5 Percentage of Sorbitol Fermenting 252 

Bifidobacteria in Feces - High Priority 
and Non-Priority Storm and Sanitary Sewage 

F-1 Geometric Mean Concentration of Fecal 267 

Indicators in Storm Sewage 

F-2 Geometric mean Concentrations of Fecal 269 

Indicators in Sanitary Sewage 

F-3 Percentage Serotypes of Pseudomonas 270 

aeruginosa in Storm and Sanitary Sewage 

F-4 Agarose gel electrophoresis of total cellular 273 

CMA from Pseudomonas aeruginosa digested with 
Sma I endonuclease 

F-5 Agarose gel electrophoresis of total cellular 275 

K^ from Pseudomonas aeruginosa digested with 
SMa I endonuclease 



APForoix 

Figure Page No. 

F-6 Agarose gel electrophoresis of total cellular 277 

CNA from Pseudomonas aeruginosa digested with 
SMa I endonuclease 

F-7 Agarose gel electropiioresis of total cellular 284 

CNA f rem Pseudomonas aeruginosa digested with 
six different endonucleases 



T.TfT T OF T ARTry; 

Table Page No. 

1 Levels of E^ coli 4 

2 Fecal Source Related to FC/FS Ratios 7 

3 Saitpling Locations of Hi<^ Priority and 11 
Non-Priority Storm Sewers, Sanitary Sewer 

and Storm Water Runoff 

4 Overall Geometric Mean Concentrations 19 
of Fecal Coliforms, E^ coli . Fecal 

Streptococci, Enterococci, Pseudomonas 
aeruginosa . Bifidobacteria and 
Clostridium perfringens Recovered from 
Sanitary Sewage as well as High Priority 
and Non-Priority Storm Sewage during Dry 
and Wet Weather Surveys 

5 Fecal Streptococci Populations Recovered 30 
from Storm Sewers and Sanitary Sewage 

during Dry Weather Survey 2, 1987 

6 Percentage of S^ faecal is Isolates Producing 32 
Acid Curd or Proteinization Reactions in 

Litmus Milk from Dry Weather Survey 1 

7 Percentage of S^ faecal is Isolates Producing 33 
Acid Curd or Proteinization Reactions in 

Litmus Milk from Dry Weather Survey 2 and 
Wet Weather Survey 1 Samples 

8 Percentage of Sorbitol Fermenting Bifidobacteria 35 
in High Priority and Non-Priority Storm Sewage, 

Sanitary Sewage and Feces 

9 Overall Geometric Mean Concentrations of Fecal 38 
Coliforms, E^ coli . Fecal Streptococci, 

Enterococci, P^ aeruginosa , and Bifidobacterium sp. 
Recovered from Sanitary Sewage as well as High 
Priority and Non-Priority Storm Sewage during Two 
Dry Weather Surveys in 1988 

10 Distribution of the Prominent Pseudomonas 41 
aeruginosa REA Patterns Among Sanitary Sewer, 

Priority and Non-Priority Storm Sewer and Storm 
Water Runoff Sairples 

11 Distribution of Pseudomonas aeruginosa REA 42 
Patterns Among Sanitary Sewer, Priority and 

Non-Priority Storm Sewer and Storm Water 
Runoff Sanples Collected during Periods of 
Wet and Dry Weather 



Taible Page No. 

12 Distribution of Streptococcus faecal is subsp. 45 
faecal is REA Patterns Among Sanitary Sewer, 

Priority and Non-Priority Storm Sewer and 
Storm Water Runoff Sanples Collected during 
Periods of Wet and Dry Weather 

13 Distribution of Streptococcus faecium subsp. 46 
casseliflavus PEA Patterns Among Sanitary 

Sewer, Priority and Non-Priority Storm Sewer 
and Storm Water Runoff Saitples Collected 
during Periods of Wet and Dry Weather 

14 Saitpling Dates for Dry and Wet Weather Surveys 60 

15 Geometric Mean Concentrations of Fecal 61 
Coliforms, Escherichia coli . Fecal 

Streptococcus, Enterococci and Pseudomonas 
aeruginosa Recovered from Hi(^ Priority Storm 
Sewage during Dry Weather Survey 1, 1986 

16 Geometric Mean Concentrations of Fecal 62 
Coliforms, Escherichia coli . Fecal 

Streptococcus, Enterococci and Pseudomonas 
aeruginosa Recovered fron Sanitary Sewage 
during Dry Weather Survey 1, 1986 

17 Geometric Mean Concentrations of Fecal 63 
Coliforms, Escherichia coli. Fecal 

Streptococcus, Enterococci and Pseudomonas 
aeruginosa . Bifidobacteria and Clostridium 
perfringens Recovered from Hi(^ Priority and 
Non-Priority Storm Sewage During Dry Weather 
Survey 2, 1987 

18 Geometric Mean Concentrations of Fecal 64 
Coliforms, Escherichia coli . Fecal 

Streptococcus, Enterococci and Pseudomonas 
aeruginosa . Bifidobacteria and Clostridium 
perfringens Recovered from Sanitary Sewage 
during Dry Weather Survey 2, 1987 

19 Geometric Mean Concentrations of Fecal 65 
Coliforms, Escherichia coli . Fecal 

Streptococcus, Enterococci and Pseudomonas 
aeruginosa . Bifidobacteria and Clostridium 
perfringens Recovered from High Priority 
and Non-Priority Storm Sewage during Dry 
Weather Survey 2, 1987 



Table Page No. 

20 Geonvstric Mean Concentrations of Fecal 66 
Coliforms, Escherichia coli, Fecal 

Streptococcus, Enterococci and Pseudomonas 
aeruginosa . Bifidobacteria and Clostridium 
perfrinqens Recovered f rem Sanitary Serfage 
during Dry Weather Survey 2, 1987 

21 Geometric Mean Concentrations of Fecal 67 
Coliforms, Escherichia coli. Fecal 

Streptococcus, Enterococci and Pseudomonas 
aeruginosa , Bifidctoacteria and Clostridium 
perfringens Recovered from Non-Priority 
Sewage and Runoffs during Wet Weather 
Surveys in 1987 

22 A Conparison of Fecal Coliforms/Fecal 68 
Streptococcus (FC/FS) Ratios (FC:FS) 

at Sairpling Sites 

23 Fecal Streptococci Populations Recovered 69 
from Storm Sewers and Sanitary Sewage during 

Dry Weather Survey 1, October 21, 1986 

24 Fecal Streptococci Populations Recovered 70 
fran Storm Sewers and Sanitary Sewage during 

Dry Weather Survey 1, October 22, 1986 

25 Fecal Streptococci Populations Recovered 71 
from Storm Sewers and Sanitary Sewage during 

Dry Weather Survey 1, October 28, 1986 

26 Fecal Streptococci Populations Recovered 72 
from Storm Sewers and Sanitary Sewage during 

Dry Weather Survey 1, November 18, 1986 

27 Fecal Streptococci Populations Recovered 73 
from Storm Sewers and Sanitary Sewage during 

Dry Weather June 10, 1987 

28 Fecal Streptococci Populations Recovered 74 
from Storm Sewers and Sanitary Sewage during 

Dry Weather June 11, 1987 

29 Fecal Streptococci Populations Recovered 75 
from Storm Sewers and Runoffs during 

Wet Weather Survey 1, July 14, 1987 

30 Fecal Streptococci Populations Recovered 76 
fron Non-Priority Storm Sewage and Runoffs 

during Wet Weather Survey, September 18, 1987 

31 Fecal Streptococci Populations Recovered 77 
from Storm Sewers (Non-Priority) and Runoffs 

during Wet Weather Survey October 21, 1987 



Table Page No. 

32 Percentage of S^ faecal is Isolates Producing 78 
Acid Curds or Proteinization Reactions in 

Litmus Milk from Dry Weather Survey 1, 
Octcber 21, 1986 

33 Percentage of S^ faecal is Isolates Producing 79 
Acid Curds or Proteinization Reactions in 

Litmus Milk frcm Dry Weather Survey 1, 
October 22, 1986 

34 Percentage of S^. faecal is Isolates Producing 80 
Acid Curds or Proteinization Reactions in 

Litmus Milk from Dry Weather Survey 1, 
October 28, 1986 

35 Percentage of S^ faecal is Isolates Producing 81 
Acid Curds or Proteinization Reactions in 

Litmus Milk from Dry Weather Survey 1, 
November 18, 1986 

36 Percentage of S^. faecal is Isolates Producing 82 
Acid Curds or Proteinization Reactions in 

Litmus Milk from Dry Weather June 10, 1987 

37 Percentage of S^ faecal is Isolates Producing 83 
Acid Curds or Proteinization Reactions in 

Litmus Milk from Dry Weather June 11, 1987 

38 Percentage of S^ faecal is Isolates Producing 84 
Acid Curds or Proteinization Reactions in 

Litmus Milk frcm Wet Weather 1, July 14, 1987 

39 Percentage of S^ faecal is Isolates Producing 85 
Acid Curds or Proteinization Reactions in 

Litmus Milk from Wet Weather Survey 
September 18, 1987 

40 Percentage of S^ faecal is Isolates Producing 86 
Acid Curds or Proteinization Reactions in 

Litmus Milk from Wet Weather Survey 
October 21, 1987 

41 Percentage of Serotypes of Pseudomonas 87 
aeruginosa from High Priority Storm Sewers 

during Dry Weather Survey October 21, 1986 

42 Percentage of Serotypes of Pseudomonas 88 
aeruginosa from High Priority Storm Sewers 

during Dry Weather Survey October 22, 1986 

43 Percentage of Serotypes of Pseudomonas 89 
aeruginosa from High Priority Storm Sewers 

and Sanitary Sewage during Dry Weather 
Survey November 18, 1986 



l^le . ■ P^ge No. 

44 Percentage of Serotypes of Pseudomonas 90 
aerijainosa from High Priority Storm Sewers 

ard Sanitary Sewage during Dry Weather 
Survey June 10, 1987 

45 Percentage of Serotypes of Pseudcanonas 91 
aeruginosa f rem Hi(^ Priority Storm Sewers 

and Sanitary Sewage during Dry Weather 
Survey June 11, 1987 

46 Percentage of Serotypes of Pseudcarxpnas 92 
aeruginosa from Uigin Priority Storm Sewers 

and Sanitary Sewage during Dry Weather 
Survey June 12, 1987 

47 Percentage of Serotypes of Pseudomonas 93 
aeruginosa f rem High Priority Storm Sewers 

and Sanitary Sewage during Dry Weather 
Survey August 17, 1987 

48 Percentage of Serotypes of Pseudomonas 94 
aeruginosa from High Priority Storm Sewers 

during Wet Weather Siirvey 1, July 14, 1987 

49 Percentage of Serotypes of Pseudomonas 95 
aeruginosa from Hi^ Priority Storm Sewers 

and Sanitary Sewage during Wet Weather 
Survey September 18, 1987 

50 Percentage of Serotypes of Pseudomonas 96 
aeruginosa from High Priority Storm Sewers 

and Sanitary Se^rage during Wet Weather 
Survey October 21, 1987 

51 Geometric Mean Concentrations of Fecal 97 
Coliforms, Escherichia coli. Fecal 

Streptococci, Enterococci, Pseudomonas 
aeruginosa , Bifidobacteria and Clostridium 
perfringens Recovered from Hi(^ Priority 
Storm Sewage during Dry Weather in 1988 

52 Geometric Mean Concentrations of Fecal 98 
Coliforms, Escherichia coli . Fecal 

Streptococci, Enterococci, Pseudomonas 
aeruginosa . Bifidobacteria and Clostridium 
perfringens Recovered from Sanitary Sewage 
during Dry Weather Survey in 1988 

53 Geometric Mean Concentrations of Fecal 99 
Coliforms, Escherichia coli . Fecal 

Streptococci, Enterococci, Pseudomonas 
aeruginosa , Bifidobacteria and Clostridium 
perfringens Recovered from Non-Priority 
Storm Sewage during Dry Weather in 1988 



Table Page No. 

54 Percentage of Serotypes of Pseudomonas 100 
aeruginosa fron High Priority Storm Sewers 

and Sanitary Sewage during Dry Weather 
Survey June 20, 1988 

55 Percentage of Serotypes of Pseudcaronas 101 
aeruginosa f rem High Priority Storm Sewers 

and Sanitary Sewage during Dry Weather 
Survey June 21, 1988 

56 Percentage of Serotypes of Pseudomonas 102 
aeruginosa from High Priority Storm Sewers 

and Sanitary Sewage during Dry Weather 
Survey June 28, 1988 

57 Percentage of Serotypes of Pseudomonas 103 
aeruginosa from High Priority Storm Sewers 

and Sanitary Sewage during Dry Weather 
Survey August 15, 1988 

58 Percentage of Serotypes of Pseudomonas 104 
aeruginosa from Hi<^ Priority Storm Sewers 

and Sanitary Sewage during Dry Weather 
Survey August 22, 1988 

59 Fecal Str^tococci Peculations Recovered 105 
from Storm Sewers and Sanitary Sewage 

during EJry Weather June 20, 1988 

60 Fecal Streptococci Populations Recovered 106 
f rcan Storm Sewers and Sanitary Sewage 

during Dry Weather June 21, 1988 

61 Fecal Str^tococci Peculations Recovered . 107 
from Storm Sewers and Sanitary Sewage 

during Dry Weather June 28, 1988 

62 Fecal Streptococci Populations Recovered 108 
from Storm Sewers and Sanitary Sewage 

during Dry Weather August 15, 1988 

63 Fecal Streptococci Populations Recovered 109 
f rem Storm Sewers and Sanitary Sewage 

during Dry Weather August 22, 1988 

64 Percentage of S^ faecal is Isolates 110 
Producing Acid Curds or Proteinization 

Reactions in Litmus Mile from Dry 
Weather June 20, 1988 

65 Percentage of S^ faecal is Isolates 111 
Producing Acid Curtis or Proteinization 

Reactions in Litmus Mile from Dry 
Weather June 21, 1988 



Table Page No. 

66 Percentage of S^ faecal is Isolates 112 
Producing Acid Curds or Proteinization 

Reactions in Litmus Mile frcm E)ry 
Weather June 28, 1988 

67 Percentage of S^ faecal is Isolates 113 
Producing Acid Curds or Proteinization 

Reactions in Litmus Mile from Dry 
Weather August 15, 1988 

68 Percentage of S^ faecal is Isolates 114 
Producing Acid Curds or Proteinization 

Reactions in Litmus Mile from Dry 
Weather August 22, 1988 

69 Summary of the Percentage of S^ faecal is 115 
Isolates Producing Acid Curds or 

Proteinization Reactions in Litmus Milk 
in 1988 Dry Weather Sanples 

APFtNOrX 

B-1 Media and Incubation Parameters for 128 

Enumeration of Selected Bacterial Groups 

B-2 Colony Forming Units per Millilitre 135 

at Room Tenperature 

B-3 Colony Forming Units per Millilitre 136 

at 15 degrees Celsius 

B-4 Effect of Chlorine at a e« of 6.0 150 

B-5 Effect of Chlorine at a pH of 9.0 150 

B-6 Effect of pH on Forms of Chlorine 160 

C-1 The Carbohydrate Fermentation Profile 184 

used to Speciate the Bifidobacteria 
Isolated from the Fecal and Sewage 
Sairples 

C-2 Gecmetric Mean Concentration of PC, 187 

E. coli and Bifidcbacteria in the storm 
and sanitary sewage samples 

C-3 Geometric Mean Concentrations of E^ coli 189 

and Bifidobacteria in the feces of humans, 
cats and dogs 

C-4 Ihe carbohydrate fermentation reactions of 190 

ATCC cultures 



Table Page No. 

C-5 The Biochemical Test Scheme used for the 191 

Identification of Bifidobacteria in the 
Profiles of ATCC Cultures and Their Gram 
Stain Characteristics 

C-6 Species of Bifidobacteria Isolated frxan the 192 

Two Selective Media that Occurs in Fecal 
and Sewage Sairples 

D-1 Source of Environmental Water used in vitro 217 

for the Study of Bif idobacterial Survival 

D-2 Conparison of Levels of Bifidobacteria 220 

Isolated from Human Feces on YN-17 and MEN 

D-3 levels of Bifidobacteria and E^ coli in 221 

Human Feces as Isolated on YN-17 and m-TEC 

D-4 levels of Sorbitol -Fermenting Bifidobacteria 222 

and Ei coli in Human Feces as Isolated on 
HBSA and m-TEC 

I>-5 Mean Survival Counts of Bifidobacteria and 223 

E. coli in Lake Ontario Water Enumerated 
Over a Maximum Three Day Period 

E-1 Ratio of Ei coli to Clostridium perfrinqens 246 

and of Bifidobacteria to E^ coli for High 
Priority and Non-Priority Storm and 
Sanitary Sewage 

E-2 Geometric Mean Concentrations of Fecal 247 

Coliforms, E. coli, Clostridium perfrinqens 
and Bifidobacteria in High Priority, 
Non-Priority Storm and Sanitary Sewage 

F-1 Media and Incubation Parameters for 262 

Enumerating Selected Bacterial Groups 

F-2 Serotype Distribution of Pseudomonas 271 

aeruginosa in Storm and Sanitary Sewage 

F-3 Result of Agarose Gel Electrophoresis of 274 

Total Cellular CMA Extracted From Pseudomonas 
aeruginosa 

F-4 Correlation of Various Serogroup Typing 280 

Schemes Based on Ag 

F-5 Worldwide Frequency of Incidence of Serotypes 282 

of Pseudomonas aeruginosa 



ABBREVIAnCNS 

REA - Restriction Endonuclease Analysis 

FC - fecal coliforms 

FS - fecal streptococcus 

P. - Pseudomonas 

B. - Bifidobacterium 

S. - Streptococcus 

E. - Escherichia 

L - litre 

IG - indoxyl-/?-D-glucoside 

SDS - sodium dodecyl sulpiiate 

EOTA - ethylenediaminetetraacetic acid 

TAE - Tris base, 1.0 sodium acetate, 0.1 M disodium EDTA 

CFU - colony forming units 

Var - variety 

ATCC - American Type Culture Collection 

E - exponential 



- 1 - 

INIRDDUCnCN 

Storm sewers were designed to channel storm water frcm urban areas into 
surface waters primarily to avoid the flooding that would occur as a 
consequence of the blocking of natural flew patterns that existed before 
urbanization. The content of storm sewers should be similar to direct runoff 
and the flew restricted primarily to storm events. A small amount of dry 
weather flow is not unusual in storm sewer lines due to ground water intrusion 
and human activities such as the watering of lawns. However, significant dry 
weather flow coi^led with high fecal coliform counts may indicate that illegal 
sanitary connections are present somev^ere in the storm sewer line. This 
situation presents a health hazard since storm sewage is not normally treated 
prior to entering receiving waters such as Lake Ontario beach areas or the 
Humber and Don Rivers which flow into Lake Ontario near beaches. 

Reports of the Toronto Area Watershed Management Study (TAWMS) in 1983 and 
1984 have identified contaminated storm sewer outfalls as a major source of 
bacterial pollution loadings to the lower Humber River watershed (Gartner Lee 
and Assoc. 1983; Weatherbee and Novak, 1984). The 1983 outfall inventory study 
of the lower Humber River basin identified ei<^ty-four dry weather outfalls 
that were considered sufficiently active to warrant intensive saitpling and 
testing (Gartner Lee eind Assoc. 1983) . A similar study initiated in the Don 
River watershed indicated that 16% of all dry weather outfalls exceeded the 
recommended guidelines for fecal coliform densities (TAWMS Tech. Rep. No. 11, 
1986) . 

The Ministry of the Environment has designated storm sewer outfalls 
discharging more than 1 L/sec. during dry weather periods and exhibiting fecal 
coliform densities of greater than 10,000 FC/lOO mL as being hi^ priority and 



- 2 - 
have called upon municipal agencies to identify and eliminate the source of the 
fecal pollution in these sewage lines. Follow up action has been initiated on 
identified outfalls in the Humber River watershed and already several illegal 
cross-connections between sanitary and storm sewers have been located and 
corrected (Weatherbee and Novak, 1984) . Despite some successful attempts, 
compliance with the Ministry's directive can be difficult at times, due to the 
fact that methods for source determination are under-developed. Escherichia 
coli v^ch is an excellent indicator of fecal contamination does not lend 
itself to source determination due to its wide-spread occurrence in all fecal 
material. Older methods such as the ratio of fecal coliforms to fecal 
streptococci (FC/FS) have been used. Hcwever, the ratios proposed by 
Geldreich in 1969 can no longer be considered valid. A major reason for this 
is that bacterial isolation and enumeration methods currently used in Ontario 
differ significantly from those used to develop the FC/FS ratio. As well, it 
has been shown that certain animal hosts; i.e. dogs, gulls and pigeons exhibit 
similar ratios to that of humans (Seyfried, Harris and Young, 1986 unpublished 
data) and that the ratios obtained from polluted waters change over time as a 
result of environmental stress (Seyfried, Harris and Young, 1986 unpublished 
data; Feachem, 1975) . 

Recently, newer methods of source determination have been proposed. These 
methods entail the use of a variety of indicator organisms as described below. 

Rationale for the Use of Selected Indicator Organisms 

The levels of pathogens in polluted water and aniinal feces are highly 
variable since the densities reflect the intestinal diseases that are prevalent 
in human or other animal populations at a given time. As a result, monitoring 



- 3 - 
of water for possible detection of waterbome pathogens requires a variety of 
conplex, time-consuming, and often insensitive procedures. The use of a 
bacterial indicator system that will detect and measure fecal pollution from 
all warm-blooded animals is a more realistic approach. 

Fecal Col i forms 

The fecal col i form bacteria do have a direct correlation with fecal 
contamination from warm-blooded animals. The ability to ferment lactose with 
gas production at 44.5°C is the principal biochemical characteristic used to 
identify fecal coliforms. Geldreich (1966) has shewn that 96.4% of the 
coliforms in human feces were positive by this test. Soils that are 
contaminated with fecal discharges or ejqxssed to polluted water will contain 
varying levels of fecal coliforms (Van Donsel, Geldreich and Clark, 1967; 
Geldreich et al . . 1968). The low levels of coliforms that have been detected 
on vegetation are derived from animal manure or ni(^t-soil used as fertilizers, 
or by contact with contaminated insects and agricultural pests (Geldreich, 
Kenner and Kabler, 1964) . During periods of rainfall, contamination that might 
be associated with vegetation could enter surface waters via storrawater 
drainage (Geldreich et al . . 1968). 

Escherichia coli 

In Europe, there is a long standing tradition to distinguish between 
total coliforms and Escherichia coli because the latter is considered to be 
more significant (Leclere et al . . 1977). According to Cabelli (1977), E. coli 
is the only coliform biotype that is consistently and exclusively associated 
with fecal wastes of warm-blooded animals. Ihus the significance of the 



_ 4 - 
presence of E. coli in surface waters is that fecal contamination due to 
humans or warm-blooded animals has occurred and therefore a potential health 
hazard risk from microbial or viral enteric pathogens does exist. The levels 
of E. coli found in various animals, birds, and sewage effluents are given in 
the table below. 

Table 1. Levels of E. coli 

(frcan Jones and White, 1983) 





Fecal production 
g/d 


Average number 
E. coli/q faeces 


Daily '. 


Load 


E. coli 


Man 


150 


13 X 10^ 


1.9 


X 


109 


Cow 


23600 


0.23 X 10^ 


5.4 


X 


109 


Hog 


2700 


3.3 X 10^ 


8.9 


X 


109 


Sheep 


1130 


16 X 10^ 


18.1 


X 


109 


Duck 


336 


33 X 10^ 


11.1 


X 


109 


Turkey 


448 


0.3 X 10^ 


0.13 


X 


109 


Chicken 


182 


1.3 X 10^ 


0.24 


X 


109 


Gull 


15.3 


131.2 X 10^ 


2 


X 


109 


Sewage 

Sewage effluent 


E. 


coli concentratior 

3.4 X 10^ - 2.8 ) 

1 X 10^ - lo' 


1 100 ml 

{ lo'^ 

7 







Freshwater 
Seawater 



E. coli survival 



mean Tgo 62.3 h 
mean Tgg 2.3 h 



Dufour's (1977) finding that E. coli showed the best relationship to 



- 5 - 
gastrointestinal illness, rather than members of the KLebsiella-Enterobacter - 
Citrobacter group, is not surprising in view of its position of dominance in 
the distribution of coliforms found in fecal wastes from humans and other warm- 
blooded animals. 

Fecal Streptococci and Enterococci 

Fecal streptococci are defined as those species of streptococci which are 
recovered from feces in significant quantities (Clausen et al . . 1977). 

The term enterococci is used to describe those species of fecal streptococci 
that grow at both 10° and 45° C and in the presence of 40% bile. Growth should 
occur at a pH of 9.6 and in the presence of 6.5% sodium chloride. Esculin 
hydrolysis is also positive for these organisms. Ihe group D enterococci 
include S. faecal is . S. faecium , S. durans . S. avium , and related biotypes 
(Sergey's Manual, 1986). 

The occurrence of fecal streptococci in water suggests fecal pollution and 
their absence indicates little or no warm-blooded animal contamination 
(Geldreich and Kenner, 1969) . Ihere is no indication that they multiply in 
natural or fecally polluted waters or soils (Clausen et al . , 1977). Cabelli 
(1977) has suggested that the enterococcus group most closely meets the 
characteristics of an ideal indicator, particularly because it survives better 
than E. coli in the aquatic environment. 

A study conducted at the University of Toronto (Seyfried, Harris and Young, 
1986 unpublished) found that differences in the relative prcportions and 
biotypes of group D str^Jtococci existed between human and animal hosts. These 
findings are also supported by previous workers (Wheater, et al. 1979, Kenner 



- 6 - 
1978; and Geldreich 1976). Hill et al. (1971) found that human subjects fed on 
a mainly mixed western diet carried a higher percentage of S. faecal is 
biotypes. Mundt (1982) also found S. faecal is to be the predominant species 
of group D streptococci in humans and was able to demonstrate marked 
differences in the litmus milk reactions of S. faecal is from humans, animals 
and plant material (Mundt, 1973) . 

Fecal Col i form to Fecal Streptococci Ratios 

The FC/FS ratio has been proposed as a means of estimating v*iether the 
pollution originated from a human or a nonhuman source (Geldreich, 1966; 
Geldreich and Kenner, 1969, Geldreich et al. 1968). Geldreich suggests that, 
"Fecal coliform bacteria are more numerous than fecal streptococci in domestic 
sewage, with a fecal coliform to fecal streptococcus ratio always greater than 
4.0. As mi^t be expected, similar ratios are common to the feces of man. 
Conversely, fecal streptococci are more numerous than fecal coliforms in the 
feces of farm animals, cats, dogs and rodents. In feces from these animals, 
the fecal coliform (FC) to fecal streptococcus (FS) ratios are less than 0.7. 
Similar lew ratios are corranon to urban stormwater and farmland drainage" 
(Geldreich, 1972). 

The obvious major weakness of this approach is that, unless the FC and FS 
die off at identical rates, the FC/FS ratio will gradually change and will no 
longer reflect the original ratio in the fresh fecal material. Geldreich and 
Kenner (1969) made note of this fact and recommended that the PC/FS ratio was 
only valid during the first 24 hours immediately following the discharge of 
bacteria into the stream. However, it is not always possible to judge the age 
of the pollution and, even if one can, one cannot always estimate the time 



- 7 - 
between excretion and discharge into the stream. As a result McFetters et 
al. (1974) concluded that the FC/FS ratio "is no longer of significance in 
determining the source of the contamination when considering bacteria that 
originate from domestic sewage" . 

IfcFetters et al . (1974) found that enterococci survive better than FC v*iich 
survive better than S. bovis and S. equinus . Feachem (1975) has prcposed that 
this differential die-away can in fact strengthen the value of the FC/FS ratio 
as a means of distinguishing human from non-human pollution. For example, in 
fecal material in v*iich enterococci are the dominant FS group (as in human 
feces) the FC/FS ratio will tend to fall viiereas in fecal material in v^ch S. 
bovis and S. equinus dominate (for instance in cattle and pig feces as 
suggested by Deibel, 1964; McFetters et al. 1974; Raibaud et al. 1961). the 
FC/FS ratio will tend to rise. A predominantly human source should exhibit an 
initially high (>4) ratio v^iich should then fall v^ereas a non-human source 
should ejdiibit an initially lew ratio (<0.7) v^ch should subsequently rise. 
Feachem 's conclusions are summarized in the folloving table. 



Table 2. Fecal source related to FC/FS ratios 



Initial 

FC/FS ratio 


Change through 
time of 
FC/FS ratio 


Probable fecal 
source 


> 4 


Rise 
Fall 


Uncertain 
Human 


< 0.7 


Rise 

Fall 


Nonhuman 
Uncertain 



- 8 - 



Pseudomonas aeruginosa 

Ihis organism is an opportunistic pathogen of man and animals v^iich may be 
spread by water. Althou(^ grcwth of the organism in water may occur under 
certain conditions, the major source of P. aeruginosa in waters appears to be 
fecal wastes of man and of animals associated with man. Studies have shown 
that there frequently appears to be little relation between populations of P. 
aeruginosa and those of other pathogens or fecal indicators (Hoadley, 1977) . 

Qiaracterization of Pseudomonas aeruginosa isolated from weiste-waters may 
also be of significant value in source determination because this species is 
found primarily in human as opposed to other domestic animal wastes; i.e. dogs 
and cats (Seyfried, Harris and Young, 1986 unpublished data; Wheater et al. 
1979) and because serotyping allows for sub-speciation of this organism into 
approximately 17 different heat-stable somatic antigenic groups (Kusama, 1978) . 
Source tracing of Pseudomonas aeruginosa infections by serotyping has been 
used successfully in many clinical trials (Young and Moody, 1974; Baltimore et 
al. 1974) . Ihe method has also been used to trace the source of infection in 
swimmers (Seyfried and Fraser, 1978) and could possibly be adapted to tracing 
studies in other environmental settings. 

Bifidobacterium spp. 

It has been suggested that Bifidobacterium spp. are good indicators of 
human fecal wastes in surface waters (Buchanan and Gibbon, 1947; Leven, 1977; 
and Resnick and Leven, 1981) . Bifidobacteria are present in concentrations of 
10^ organisms per gram of feces in humans (Geldreich, 1979) but have a very 



_ 9 _ 

limited distribution among other animals (Mara and Oragui, 1983) . Ihey have 
also been recovered from raw sewage (Resnick and Leven, 1981) . Mara and 
Oragui (1983) reported that sorfeitol fermenting species of bifidobacteria were 
exclusive to human fecal wastes and proposed a membrane filtration method for 
recovering bifidobacteria from surface waters. 

Clostridium perfringens 

Clostridium perfringens was suggested as an indicator of the pollution of 
water with fecal wastes in the late 1890s (Cabelli, 1977) . However, because of 
the organism's extreme resistance, abundance in deconposing organic matter and 
soil, and failure to occur in numbers that correlate with the results of a 
sanitary survey (Levine, 1921) it is rarely used as an indicator. Nonetheless, 
the enumeration of C. perfringens spores as a water guality indicator appears 
to have specific and limited applications, primarily as an adjunct to the 
commonly used coliform and fecal streptococcus indicators. 

C. perfringens is an indicator of choice v*ien the requirement is for 
measuring remote and intermittent sources of pollution reaching an area (such 
as, tracing sewage sludge dunped into bodies of water or the infiltration of 
ground water supplies with fecal wastes) and in instances where other microbial 
indicators are rapidly destroyed, such as chlorinated water supplies. 

Scope ', .. • 

In order to determine the relative merits of the novel source 
determination methods and to devise a methodology for the detection of human 
fecal wastes in storm sewer lines, a study to characterize the bacterial 
populations found in urban storm and sanitary wastes was initiated in the fall 



- 10 - 

of 1986. Fecal indicator bacteria were enumerated in sanitary, storm and 

priority storm sewage and storm water runoff. Fecal streptococci and 

Pseudomonas aeruginosa were isolated and identified according to the 
phenotypes, serotypes and genotypes present in the different sairple types. 

Bifidobacteria were also enumerated fron storm and sanitary wastes and 
recovered isolates were tested for sorbitol fermentation. 

MEmODS 

Sampling Sites 

Sites A, B and C (shown in Table 3 and Fig. 1) in the Mount Steven Trunk 
storm sewer line were sampled because this area was designated a high priority 
sewer by the Ministry of the Environment. The non-priority sites, selected for 
conparison, were X, Y and Z in the Mount Steven Trunk storm sewer branch lines. 
Curing periods of wet weather, storm water run-off was also collected at X, Y 
and Z sites. These sairples were labelled R, G. and Q, respectively. Saitples 
D, E and F were obtained frcm a sanitary sewer in close proximity to the 
priority storm sewer sanpling points. 

Sample Collection 

IXjring periods of dry weather, triplicate sanples were collected from each 
sampling point in the sewers over a two to four-day period. The dry weather 
sanples were obtained in October, 1986; June, 1987; August, 1987; June, 1988; 
and August, 1988. Wet weather sanples were collected from the sewers and the 
street run-off during rainy days in July, September and October, 1987. (See 
Appendix Table 14 for precise sampling dates) . 



- 11 - 



Table 3. Saiipling locations of high priority and non-priority 
storm sewers, sanitary sewer and storm water runoff. 



Sample 
Description 



Code 



Site 



High Priority 
Storm Sewer Line, 
Mount Steven 
Storm Sewer Trunk 



B 



Danf orth and Jones Avenues - furthest 
in-line sanpling point (near source of 
suspected solution input) . 



Pape and Strathcona 
saitpling point) . 



Avenues (mid-line 



First and Broadview Avenues (near outfall) 



Non-Priority 
Storm Sewer 
Branch Lines 



Chatham and Jones Avenues (connects to main 
line above saitpling point B) . 



Danf orth and Woody crest Avenues (connects to 
main line above sanpling point A) . 



Pape and Cavell Avenues. 



Storm Water 
Runoff 



G 
R 
Q 



Chatham and Jones. 

Danf orth and Woodycrest Avenues 

Pape and Cavell Avenues 



Sanitary 
Sewage Line 



D 
E 
F 



Danf orth and Jones Avenues 
Strathcona and Pape Avenues 
First and Broadview Avenues 



- 12 - 



o 



<r 



CQ 



LU 



N 



O 



I 

I 
1 
I 



LL 







U1 
01 






c 






^— 






•— 






s- 






(U 






s 






lU 






in 






> 






u 




LLI 






z 


c 


1 1 


1 ^^ 


lO 


z ^ 


U1 

-3 




J cc 




^ 


^ LU 


u 




en 


^0 


» LLI 


E 


c 


» CO 


O 


S s 


4^ 

C 

> 


i g 


0) 


O 2 


c 


H < 


CO CO 


o 






E 






IS 






5) 






lO 






^ 






u 






^— 






■^ 






2 






c 






Ol 






"G 






1 



o 



- 13 - 
The saiiples were collected in sterile glass containers, transported to the 
laboratory on ice, and processed within 6 hours of collection. 

Fecal specimens fran both humans and amirals were also obtained. 

Bacterial Isolation and Enumeration 

Analysis of the sairples for indicator bacteria was by membrane filtration 
of appropriate dilutions of the saitples through Gelman GN6 47 ram cellulose 
nitrate filters with a pore size of 0.45 um (Standard Methods, 1985). The 
filters were planted on media appropriate for the recovery of the various 
indicator bacteria. 

Fecal coliform bacterial densities were determined by planting the filters 
on m-TEC agar (Dufour et al. 1981) and incubating for 23 ± 1 hours at 44.5 + 
0.5° C. Both target and non-target colonies were counted. Target colonies 
were yellcw, yellcw-green and yellcw-brcwn; non-target colonies were blue to 
blue-green in colour. To ensure the accuracy of the counts, only results 
obtained from filters with target counts between 10 and 100 colonies were used 
to calculate the bacterial density per 100 mL of water sanple. 

A second step for the determination of E. coli by urease treatment (Cufour 
and Cabelli, 1975) was incorporated into the m-TEC procedure. Filters with 
appropriate target counts (i.e. between 10 and 100 target colonies) were 
removed from the m-TEC plates and placed on filter pads soaked in a urea phenol 
red solution (Dufour and Cabelli, 1975) . The filters remained in contact with 
the filter pad for 15 min. to allcw for deaminization by non -E. coli coliform 
bacteria processing urease. A second count of all urease-negative colonies 
(all yellow, yellcw-green and yellcw-brown colonies) was taken. 

Fecal streptococci determinations were made by planting the filters on m- 



- 14 - 
Enteroccxxnjs agar (Difcx>) (AHiA, 1985) . The medium was incubated for 48 hr. 
at 35 °C and a ccfunt of all pink to purple target colonies taken. Upper and 
Icwer counting limits of 10 to 150 target organisms were applied to the 
reported results. 

Enterococci were recovered on m-ME agar (AEHA, 1985) v*iich also contains 
indoxyl-B-D-glucoside (IG) . The m-ME plates were incubated for 48 hours at 
41.5 ± 0.5° C. The incubation time was modified from the original 24 hr. 
suggested by Dufour as it allowed for a slight increase in recovery of target 
organisms. Ihe addition of IG to the medium facilitates differentiation of the 
B-D-glucosidase enterococci from other fecal streptococci. Both target and 
non-target colonies were enumerated. Targeted colonies on m-^1E were purple, 
v*iite-blue to dark blue with blue haloes from degradation of the IG. Non- 
targets were pink to maroon non-haloed colonies. Counting range limits of 10 
and 150 were also applied to these results. 

Pseudomonas aeruginosa densities were determined using m-PA agar (AHiA, 
1985) . The medium was incubated at 41.5 + 0.5° C for 48 hr. and a count of 
all flat spreading brcwnish-green or tan colonies obtained. Upper and lower 
counting limits of 10 and 150 were afplied to the results. 

Bi f idobacter ium spp. were isolated on the YN-17 medium described by Mara 
and Oragui (1983) . Target bifidobacteria colonies appear dark blue to black. 
Greenish coloured colonies were not counted; however, to ensure that all 
bifidobacteria were enumerated, crystal violet stains were made of colonies 
which had a different size or colour. Adjustments to the count were made if 
necessary. 

Clostridium perfrinaens was isolated using a medium originally developed 
by Bisson and Cabelli (1979) and modified by the Ministry of the Environment 



- 15 - 
Southeastern Region Laboratory (in-CP2) (M.O.E., 1986). The sanples were 
pretreated at 70° C for 15 or 30 min. to destroy the vegetative cells. 
Appropriate volumes of the sairples were passed thrtxK^ membrane filters and the 
filters were placed on m-CP2 plates. The plates were then incubated 
anaerobically at 37° C for 48 hr. Target colonies appear yellcw with a large 
black centre v*iich can extend fairly close to the circumference; the colonies 
do not possess a blue halo. Nontarget colonies have a blue halo and they can 
be yellow or yellcw with a black centre. 

Formulations for the aforementioned media are provided in the Appendix. 

All bacteria were identified to the species level using standard taxonomic 
methods. 

Bacterial Characterization . . .. 

Approximately 2,500 fecal streptococci were recovered on m-Enterococcus 
agar and m-ME agar during the dry and wet weather surveys. The isolates that 
were identified as varieties of S. faecal is were tested for their reaction in 
litmus milk broth (Difco) using the method of Mundt (1973) . 

Serotyping of the P. aeruginosa isolates was carried out using a 
Pseudomonas Antisera Kit (Difco) . The organisms were sub-speciated into the 17 
different heat-stable somatic antigen groips described by Kusama (1978) . 

Genotypinq 

For the restriction enzyme analysis (REA) of P. aeruginosa , total cellular 
raJA was extracted using a method described by Bradbury et al. (1984, 1985) . A 
1.5 mL volume of an 18 hour nutrient broth culture inoculated with P. 
aeruginosa was transferred into an Eppendorf tube and centrifuged in a 



- 16 - 
Microfuge 12 (Beckman) for 3 minutes at 7500 x g. The supernatant was 
discarded and the pellet loosened by vortexing. A 291 uL volume of PEB I 
buffer containing 10 mg/mL lysozyme was added and the mixture incubated for 20 
minutes at 35° C. A 9 uL amount of 5M NaCl was added, thorou^ily mixed, and 
then 150 uL of 10% sodium dodecyl sulfate (SDS) was added. The solution was 
gently mixed and incubated for 10 minutes at 37° C. Following the addition of 
450 uL phenol: chloroform risoarayl (25:24:1), the mixture was vortexed and 
centrifuged at 7500 x g for six minutes at room tenperature. The upper aqueous 
phase was removed with a pasteur pipette and transferred to an Eppendorf tube. 
One mL of 95% cold ethanol was added, the tubes vigorously shaken and stored at 
-20° C ovemi(^t. The mixture was centrifuged for 3 minutes at 12, 000 x g, 
the si.;pematant discarded and the pellet redissolved in 250 mL CNA wash buffer. 
A total of 500 uL of 95% cold ethanol was added, the mixture stored at -20° C 
for 20 minutes, and centrifuged at 1,200 x g for 3 minutes. The supernatant 
was discarded and the pellet allowed to dry at 37° C for 10 minutes. The 
pellet was dissolved in 100 uL of distilled water and stored at 4° C until 
digested. 

Restriction digests were performed using Sma I according to the 
manufacturer's instructions (Boerhinger Mannheim). A 10 uL aliquot of double 
strength (2X) Sma I buffer was placed in an Eppendorf tube and 10 uL extracted 
CWA added. A 2 uL sanple of Sma I enzyme was added and the mixture was 
incubated for 1 hour at 37° C to allow for ccsiplete digestion. Following the 
addition of 1 uL of 0.15M EETA + 0.4 mg/mL RNase A, the tube was incubated at 
37° C for 20 minutes. A 5 uL volume of 5X sairple buffer was then added to the 
restriction digest. Saitples were electroj±ioresed on 0.7% agarose gel for 16 
hours at 27 volts. Gels were stained with 1 mg/mL ethidium bromide in IX TAE 



- 17 - 
(Tris base, 1.0 sodium acetate, 0.1 M disodium EDIA) for 1 hour and destained 
for 2 hours in distilled water. Hiotography was done using U.V. lii^t at 300 
nm and a red No. 23A polaroid 66p/N film with an ejqxjsure time of 30 seconds. 

Fecal streptococci were grcwn in Brain Heart Infusion broth (Difco) at 37° 
C for 4 hours and the total cellular CNA extracted as described previcJusly. 
Restriction digests were performed using Bam HI enzyme according to the 
manufacturer's directions (Boehringer Mannheim) along with a 10 liL aliquot of 
2X Bam HI buffer. 

Bifidobacterium sp. were grcwn anaercbically in MRS broth (Oxoid) for 48 
hours at 37° C. The same method as was used for P. aeruginosa and fecal 
streptococci was enployed to extract total cellular DMA from Bifidotecterium 
spp. When difficulties were experienced with the cellular DNA extraction 
procedure, the follcwing treatments were added: 

1. hi^ concentration of lysozyme (i.e. 20 mg/mL) ; 

2. sodium dodecyl sulfate (SDS) with normal lysozyme concentration; 

3. alkaline lysis; 

4. SDS with proteinase K; 

5. proteinase K without SDS; 

6. Triton X-100; 

7. 4M guanidine isothiocynate ; and 

8 . lysostaphase . 

Three different enzymes, Sma I, Bam HI and Cfo, with their appropriate 
buffers, were used for the restriction enzyme analysis of the bifidobacteria. 



- 18 - 
RESUIJS AND DISCUSSICN 

1986 and 198 7 Surveys 

Ihe concentrations of fecal indicator bacteria and Pseudcmionas aeruginosa 
found in sanitary s«^rage, hi<^ priority and non-priority storm sewage, and 
storm water runoff are presented in Tables 15 to 21 (Appendix) . The results 
summarized in Table 4 shew that the concentrations of fecal indicator bacteria 
in the Mount Steven Storm Trunk were hi<^ during both dry weather surveys. 
Although levels of greater than 10,000 fecal coliforms per 100 mL occurred 
throughout the main line (saitpling points A, B, and C) high densities were 
noted most frequently at sites A and B. The source of the suspected fecal 
contamination is thought to be near site A, but the hi(^ fecal coliform and E. 
coli levels exhibited in sanple B suggest that a second contaminant input may 
occur somewhere in-line between points A and B. The concentrations of fecal 
coliforms, and more specifically of E^ coli . in saiiple C (near the outfall) 
tended to fluctuate but were usually Icwer than at sites A ard B. In 
conparison, as shewn in Figs. 2 and 3, the concentrations of fecal indicator 
bacteria in sanitary sewage were consistently high, as would be expected in 
this type of sarrple. 

As nay be seen in Table 22 (Appendix) , the fecal coliform to fecal 
streptococcus ratios observed in the sanitary sewage sanples in dry weather 
were generally above 4, indicating human fecal input. Ihe FC/FS ratios varied 
from day-to-day in the storm sewer line, but there was a tendency tcward ratios 
of > 4.0 at points A and B. At site C, the FC/FS ratios were always below 4 
which would suggest that no source of contamination is present and that the 
area is being iitpacted upon by upstream pollution. 



- 19 - 






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- 22 - 

Pseudcmonas aeruginosa was also present in high concentrations in the 
sanitary sewer (Fig. 4) . Althoijgh this organism was found to occur in only 12% 
of the human population (Sutter et al . . 1967), it is consistently isolated from 
sewage and highly polluted surface waters (Bonde, 1963; Hoadley, 1967; Cabelli, 
Kennedy and Levin, 1976) . 

Pseudomonas aeruginosa was recovered from the high priority storm sewer 
(A, B and C) during all dry weather surveys in 1986 and 1987 (Tables 15 to 20 
Appendix) . The levels ejdiibited during the second dry weather survey were 
hi^er than those recovered in survey 1 (Table 4) possibly due to the fact that 
the second survey was conducted during the summer period as opposed to the late 
fall sairpling of survey 1. Pseudomonas has been shown to exhibit regrowth in 
nutrient enriched waters at hi^ tenperatures (Hoadley, 1977) . 

Previous investigators have shewn P^. aeruginosa to be more indicative of 
human rather than animal fecal wastes (Wheater et al . , 1978, 1979). The 
concentrations present at Points A and B, particularly during dry weather 
survey 2, would suggest that the contaminate irput at these sites is human in 
origin. Levels of Pseudomonas at point C tended to be lower than at points A 
and B, again confirming that the source of the contamination lies i^sstream of 
this point. 

Pseudomonas aeruginosa was not isolated from the high priority storm sewer 
during the first dry weather survey on October 28. Rainfall on the previous 
evening may have caused dilution effect in the sewer such that P^ aeruginosa 
could not be detected. Any incoming fecal material from street runoff would be 
of non-human origin (e.g. dog) (Geldreich, 1979) . Ihis would cause an increase 
in the fecal coliform, E^ coli and fecal streptococcus concentrations in the 
sewer but would not necessarily increase P^. aeruginosa concentrations since 



- 23 - 



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- 24 - 
animal feces (i.e. dcgs, cats, raccoons) generally do not contain Pseudomonas 
(Seyfried, Harris and Young, manuscript in pr^>aration) . 

Bifidobacteria was recovered frcm both hi^ priority storm and sanitary 
sewage during the dry weather surveys (Tables 15 to 20, Appendix) . Ihis 
organism is thought to occur primarily in human wastes (Mara and Oragui, 1983) 
and was found in concentrations of 10^ to 10-^° in the human feces analyzed as a 
separate facet in this study. The hi^ concentrations of this bacterium in the 
storm sewer at points A and B and the Icwer concentrations found at point C 
(Fig. 5) again confirms that human fecal input occurs at the two upstream 
points because this organism exhibits rapid die-off in surface waters (Oragui, 
1982) . . 

Clostridium perfringens was included as a parameter during the second dry 
weather survey. Current literature available on the bacterium suggests that it 
is associated with both human and animal wastes (Bisson and Cabelli, 1980; 
Geldreich, 1979) . Generally the organism is regarded as a good indicator of 
fecal pollution in situations v*iere there has been environmental stress due to 
disinfection, prolonged transit time or the presence of toxic wastes (Bisson 
and Cabelli, 1980; Geldreich, 1979; Fujioka and Shizuraura, 1985). C^ 
perfringens was recovered from both high priority storm and sanitary sewage, 
and was present in higher concentrations in sanitary sewage (Fig. 6) although 
it could not be isolated from the human fecal sanples assayed. According to 
Geldreich (1979) , C^ perfringens is found in concentrations of 10^ to 10^ per 
gram of human feces but is only present in 13 to 35 percent of the population. 

Additional sairples X and Y analyzed during the second dry weather survey 
were collected from branch lines connecting to the main trunk line (see Table 3 
and Fig. 1) . Ihese lines were thought to be non-priority storm sewage 



- 25 - 



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- 27 - 

according to the City of Toronto Public Works Department. Sairple X e>diibited 
low fecal indicator counts as would be ej^^ected in non-priority storm sewage. 
The sanple also contained very lew Pseudcanonas and Bifidobacterium levels 
v^iich woiild indicate the absence of human feccil input at this site. Sample Y, 
however, e>±Libited hi(^ concentrations of fecal col i forms and E^ coli , and on 
June 12 the FC counts were greater than 10,000/100 mL. Concentrations of 
Pseudomonas aeruginosa and Bi f idobacter ium were also high in this sairple 
suggesting that a contaminant irput, possibly of human origin is impacting on 
the sewer at or near this site. Although site Y is located upstream of site B, 
(see Fig. 1) it cannot account for the magnitude of pollution exhibited at B 
since the bacterial concentrations recovered at Y were lower than those 
presentat the B site. Because of its high fecal indicator bacterial levels, 
sanple Y was groi53ed with the high priority storm sewage. 

The FC/Fs ratios exhibited in sattples X and Y were generally belcw 4. 
This would appear to contradict the evidence of human fecal input at site Y, 
i.e. Bifidobacteria concentrations. Previous studies however, have shown that 
FC/FS ratios tend to fluctuate over time as a result of the different die-off 
rates of the two bacterial grotps (McNeil, 1985). It may be that the fecal 
pollution irput inpacting on the sewer at site Y is located in-line above this 
sanpling site. Since Bifidobacteria also tend to die-off rapidly (Oragui, 
1982) , it would be interesting to determine if samples taken above site Y in 
this branch line exhibited an increase in this organism cis well as an increase 
in the FC/FS ration and levels of other fecal indicator bacteria. 

Fecal indicator bacterial concentrations recovered from non-priority storm 
sewage and storm water runoff during wet weather (Table 4) revealed that 
street runoff is heavily contaminated with fecal material and contributed 



- 28 - 
greatly to the cnntamination in storm sewer lines during storm events. The 
rainfall for this event commenced on the afternoon of July 13 and continued 
until the late morning of July 14. Sanples Q, X and Z were collected after the 
first flush had occurred. Sanple P was collected from street water runoff at 
Parliament and Carlton streets at the start of the rainfall event and this may 
account for the higher Pseudomonas levels exhibited in this storm water runoff 
sanple over that of storm water sanple Q. As well, the fact that sample P was 
collected from • a different geographical location may account for this 
difference . 

Allen Gardens is an area v4iere gulls and pigeons feed and both of these 
bird species were found to carry Pseudomonas (Sey fried, Harris and Young, 1986, 
unpublished data) . During wet weather, Pseudomonas aeruginosa concentrations 
in storm water and storm sewage may cLLso be increased by runoff frcm vegetation 
(Hoadley, 1977) . 

The fecal coliform to fecal streptococci ratios eidiibited in storm water 
and storm sewage during wet survey 1, were all below 4,0 suggesting fecal input 
of non-human origin. It would appear that FC/FS ratios may still be a useful 
diagnostic tool for source determination in storm sewer lines when applied 
close to the irput, although many investigators cautioned its use in surface 
water analyses (Wheater et al . . 1979; Palmer 1984; Diebel 1964). 

Bifidobacteria were recovered from non-priority storm sewage samples X and 
Z during wet weather. Concentrations exhibited in sanple X were much higher 
than those recovered during dry weather at this site. Our studies shewed that 
animals such as dogs can carry Bifidobacteria in their feces. 

The distribution of fecal streptococcal populations recovered from storm 
and sanitary sewage during the dry and wet weather surveys is given in Tables 
23 to 31 (Appendix) . The results from dry weather survey 2 are summarized in 



- 29 - 
Table 5. Streptococcus faecium comprised the greatest percentage of the fecal 
streptococcal group found in storm sewage and sanitary sewage during dry 
weather with the exception mainly of sairples B arri F on a few sairpling days. 
Seyfried, Harris and Young (1986 unpublished data) found S^ faecium to be 
predominant in human fecal saitples as well as several animal species (i.e. 
dogs, raccoons). Studies by Wheater et al . (1979) have also shown S^ faecium 
to predominate in human feces and sanitary sewage. However, other workers 

(Cooper and Ramadan, 1955; Kenner, 1978; Kjellander, 1960; and Mundt, 1982) 
have found a greater percentage of S^ faecal is varieties. Dietary differences 
have been shown to account for variations in the streptococcal flora of the 
human intestinal tract (Hill et al . . 1971; Finegold et al . . 1975). There is a 
tendency for S^ faecium to survive for longer periods than S^ faecal is in 
polluted waters (Dufour 1985 personal communication) which may account for its 
greater recovery from storm and sanitary sewage. Within the S^ faecal is group 
there was some variation in recovery of S^. faecal is yar. faecal is and S. 
faecal is var. liquefaciens in the storm and sanitary sewage saitples, with both 
varieties predominating during different dry weather surveys and at different 
sanple points in both sewers. Other workers have found S^ faecal is var. 
faecal is to be dcminant in human feces (Kenner, 1978) . Geldreich (1979) found 
that Si faecal is var. liquefaciens was present in only 26 percent of the human 
population and could be recovered from environmental sources. Site X (non- 
priority) ejdiibited lew percentage of Sj. faecal is varieties and showed a hi(^er 
percentage of S^ faecium var. casseliflavus an organism which is found on 
vegetation (Mundt and Graham, 1968) and in the feces of animals such as geese 

(Seyfried, Harris and Young, 1986, unpublished data) . S^ faecium var. 
casseliflavus was also recovered in high percentages in storm water and storm 



- 30 - 



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sewage cJuring wet weather but was generally not recovered from sanitary sewage 
and has not been shown to occur in human feces (Seyfried, Harris, and Young, 
1986 unpublished data) . Other str^Dtococci species not recovered in sanitary 
sewage but found in storm sewage were S^ bovis and S^ avium . Both of these 
species are fourd in animals such as dogs but not in human fecal material 
(Seyfried, Harris and Young, 1986 unpublished data) . 

The litmus milk reactions of S^ faecal is varieties summarized in Tables 
6 and 7 (from Tables 32 to 40, Appendix) demonstrate that acid curd producing 
strains are common in sanitary sewage. Mundt (1973) found that over 90% of S^ 
faecal is cultures from non-human sources such as animals, plants and insects 
gave proteinization reactions in litmus milk, v^iile isolates from human feces 
produced an acid curd. Seyfried et al . (unpublished data) also found that 
isolates of S^ faecal is from humans did not proteinize litmus milk. However, 
these same isolates also did not demonstrate the ability to produce acid curd 
reactions . 

Most of the isolates from sanitary sewage giving proteinization reactions 
were S^ faecal is var. liquefaciens and were probably of non-human origin 
because this variety can occur on such environmental sources as plants (Mundt 
et al . . 1959). Isolates from high priority storm sewage shewed a hi(^ 
percentage of acid curd production, but this reaction was also given by some of 
the non-priority storm sewage isolates from sanple X during dry and wet weather 
and by isolates obtained from storm water sample Q. Although storm water and 
non-priority storm sewage S^ faecal is isolates will have to be tested before 
the usefulness of this test can be assessed, it would appear that the litmus 
milk reactions are too nonspecific to be of value. 



- 32 - 



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The greatest percentage of Pseudoroonas aeruginosa serotypes in hi^ 
priority and non-priority storm sewage, sanitary sewage and storm water runoff 
were serotype number 6 (Tables 41 to 50, Appendix) . Other serotypes comnvon to 
all sanple types were serotypes 1, 10, 11, 4, 3, and 2. Previous research has 
shewn roost of these isolates to be common to both humans and animals (Habs 
1957; Sandvik 1960; and Verder and Evans 1961). On occasion, serotype 10 was 
found to be more prevalent in storm sewer sample Y than was serotype 6. This 
serotype was rarely isolated from non-priority storm sewage and storm water 
runoff and was isolated from sanitary sewage but not to the degree that would 
be expected if this serotype was coiranon to fecal material. Overall, the value 
of Pseudomonas serotyping as a means of pollution source differentiation 
appears to be limited and perhaps it would be more useful to look at 
Pseudomonas concentrations in storm sewage. 

The percentage of sorbitol fermenting Bifidobacteria in human feces and 
sanitary sewage is high (Table 8). Mara and Oragui (1983) have found sorbitol 
fermenting species of Bifidotacteria, i.e. B^. adolescentis and B^ breve to be 
exclusive to human feces. Other workers (McNeil 1985) have also reported this 
fact. The presence of these organisms in human feces is not affected by diet 
and geographical variation (Drasor 1974) as are group D streptococci. 

Bifidobacteria isolates obtained from dog and cat feces did not ferment 
sorbitol. Although some of the non-priority storm sewage isolates gave 
sorbitol fermentation reactions not enough isolates were tested (i.e. saitple 
X) to make any definite conclusions about the results. Isolates from high 
priority storm sewage samples A and B showed somev>^iat higher percentages of 
sorbitol fermentation reactions. Sanple Y, v*iich was originally submitted as 
non-priority storm sewage showed a hii^er percentage of the sorbitol 



- 35 - 

T^tole 8 

Percentage of Scaijitol Fermenting Bifidabacteria in Hi^ Priority 
and Non-Priority Storm Sewage, Sanitary Sewage and Feoes 



Sanple 



Souroe 



Total Isolates 
Identified 



Number of 

Sorbitol 

Fermentors 



D,E,F 


Sanitary Sewage 


A 


Hi(^ Priority 


B 


Storm Sewage 


C 




Y 




X 


Non-Priority 


Z 


Storm Sevage 


Feces 


Human 




Dog 




Cat 



37 
35 
29 
12 
32 
15 
5 
38 
24 
18 



24(65) 
8(23) 
4(14) 


13(41) 
3(20) 
2(40) 

23(61) 





( ) percentage 



- 36 - 
fermenting strains than hi^ priroity storm sewage samples A and B and as 
previously mentioned, this site could possibly be inpacted on by a human 
sanitary input. 

It would appear, based on these results, that sortiitol fermenting 
Bifidobacteria may be good indicators of human fecal contamination. 

SUmARY 

Summary of 1986-1987 Survey Results 

1. Fecal contamination, most likely of human origin, is present in the hi^ 
priority storm sewer line at or near sampling points A, B and Y. 

2. Upstream contamination is impacting on the storm sewer at the downstream 
area C. 

3 . Street runoff during wet weather is highly contaminated with fecal 
material. 

4. Bifidobacteria may be useful as indicators of human fecal wastes in storm 
sewage. 

5. A high percentage of acid curd producing strains of S^ faecal is may be 
indicative of sanitary wastes in storm sewer lines but the results are 
inconclusive. 

6. FC/FS ratios may be useful as a supplementary interpretive tool for source 
differentiation within storm sewer lines. Ihis would have to be 
investigated further because the results were not consistent at all 
points. 

7. Hi(^ Pseudomonas aeruginosa concentrations in storm sewage may indicate 
the presence of human sanitary wastes but Pseudomonas serotyping is not 
applicable to source differentiation. 

8. Source determination during storm events cannot be acconplished with a 



- 37 - 
hi^ degree of accuracy. 

1988 Surveys 

Data frcm the 1986-1987 surveys (Seyfried et al . . 1987) suggested that 
fecal contamination, possibly human in origin, was evident in the storm sewer 
line near sites A and Y. The results of the 1988 surveys, presented in Tables 
51 to 53 (Appendix) and summarized in Table 9, add support to this conclusion. 
As may be seen in the table, the fecal coliform levels in the hi^-priority 
storm sewer were greater than 10,000/100 mL at site A in June and at all sites 
in August. It should be noted that counts of all indicator organisms tended to 
be hi^er in August, possibly due to regrcwth of the bacteria in the warmer 
nutrient-enriched waters (Hoadley, 1977) . 

As mi^t be expected, fecal coliform and fecal streptococcus counts were 
highest in sanitary sewage and the fecal coliform to fecal streptococcus ratio 
was greater than 4 in these sairples (see Table 22, ^pendix) . Although a ratio 
greater than 4 was observed at site A in the priority storm sewer, at other 
storm sewer sites the FC/FS ratios were generally belcw 4 suggesting that there 
was little or no human fecal input (Geldreich and Kenner, 1969) . 

P. aeruginosa and Bi f idobacterium sp. were shown in the 1987 study to have 
potential as indicators of human fecal waste. Collaborative data presented in 
Table 9 shows that counts of both organisms were higher in sanitary and 
priority storm sewage than in non-priority storm sewage. 

Mara and Oragui first proposed the use of sorbitol fermenting 
bifidobacteria as indicators of human fecal pollution in 1983. Kator and 
Rhodes (1988) also used these organisms to differentiate human from animal 
sources of pollution in shellfish growing waters. The species of 
Bifidobacterium that are reportedly human specific and sorbitol fermenting are 



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B. adolescentis and B^ breve . In this study we were able to isolate B^ breve 
and Bj_ bifidum from human fecal material ; however, we also recovered B^ adolescentis 
from dog fecal samples and B^. breve . B. miniinum and B^ thermophilum from chicken 
feces. Twenty-one isolates that were thought to be Bi f idobacter ium on the 
basis of their morphology were recovered from eight different sewage samples. 
Of the 21 isolates, only two could be identified by biochemical testing. The 
two were found in the non-priority storm sewer at site Z and were classified as 
mannose + and mannose - strains of B^ thermophilum . 

Based upon our prior use of restriction enzyme analysis to distinguish 
between different strains of Klebsiella pneumoniae (Seyfried et al . . 1989), it 
was felt that genotyping might assist in determining the source of the Bifidobac- 
terium strains under investigation. Three enzymes were used to digest v/hole 
cell IXIA from B^ adolescentis and two Bif idobacter ium sp. isolated from chicken 
feces. Restriction (REA) patterns using the total cellular n^ restriction 
enzyme analysis were obtained. However, \jhen we attempted to repeat the restriction 
enzyme analysis, using the three different enzymes and eight modifications of 
the procedure (as described in the Methods section) , we were unable to obtain 
satisfactory REA patterns. It is probable that isolates from the YN-17 medium, 
thought to be Bi f idobacter ium sp. , were actually streptococci because of the 
initial confusion concerning the colony description (see Discussion in the 
report by In-ja Huh in the Appendix) . 

Similar to the 1986 and 1987 survey results, serotyping of the P. aeruginosa 
1988 survey isolates showed that serotype 6 predominated in the priority and 
non-priority storm sewage, sanitary sewage and storm water runoff (Tables 54 to 
58, Appendix). Serotypes 1, 10, 11, 4, 3 and 2, although not as prevalent as 
6, were also common in all categories of samples. 



- 40 - 

Seventy-ei(^t strains of P^ aeruginosa frcm the sanitary sewer and 112 
strains recovered from the storm sewer and storm water runoff were also 
genotyped. Forty-six different REA patterns were noted among the 191 isolates. 
As may be seen from Table 10, there was an interesting distribution of patterns 
among the sample groi^js. For exanple, REA patterns 1', 6', 6' and 13' were 
found among isolates from the three sampling sites in the sanitary sewer. The 
fact that these same patterns or genotypes were also prevalent in the priority 
storm sewer samples suggests that they may be typical of himian fecal isolates. 
A comparison of the corresponding serotypes for each genotype showed that the 
serotypes tended to be widely distributed. For exaitple, the 1' genotype was 
found in serotypses 1, 6 and 10; REA pattern 6 was distributed among serotypes 
1, 3, 4, 6 and 11; 6' occurred in serotypes 1, 6, 9, 10 and 11; and the 13' 
genotype was found in serotypes 6 and 10. Because it seemed lonusual to find 
such a hi(^ number of genotype 6 isolates in the non-priority storm and runoff 
saitples, the source of these organisms was examined. It was found that the 
bacteria were all isolated during the wet weather sampling in Jvily, 1987 from 
sites X and P. Ihe P site was an additional street runoff sample taken at the 
beginning of the rainfall event. Ihese isolates, belonging to REA pattern 6, 
were evenly divided between seroytpes 1 and 6. 

Additional information provided in Table 11 shows that genotypes 1, 2, 4, 
7, 9, 11", 12 and 14 were all isolated from sanitary and priority storm sewers 
and not from non-priority storm sewers or storm water runoff. 

In conparison P. aeruginosa isolates with genotypes 18 and 20 were found 
solely in the non-priority sewer and storm runoff sairples. The REA pattern 18 
organisms were distributed between serotypes 3 and 6, v*iereas pattern 20 was 
found in serotypes 1 and 3. Serotype 3, REA pattern 18 P^ aeruginosa isolates 



- 41 - 











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- 44 - 
were found in the storm water runoff sanples at site Q, and in the sewer that 
collected this same storm water at site Z. Similarly, REA pattern 20 isolates 
were recovered frcan water runoff at site R and in the receiving storm sewer at 
site X. The fact that the genotype 20 organisms belonged to both serotypes 1 
and 3 suggests that genotyping is probably a more concise method of 
fingerprinting P^ aeruginosa than serotyping. From the results, genotyping 
appears to be a promising method of tracing P^ aeruginosa from human and animal 
sources. 

Similar to the 1986-87 streptococcal results, acid curd production was 
observed among the sanitary sewage and high priority storm sewage S^ faecal is 
isolates (Table 64 to 69, Appendix) . However, there did not appear to be a 
marked difference between the aforementioned isolates and those from the non- 
priority storm sewers. All S^ faecal is isolates tended to produce a variety of 
reactions in litmus milk. 

Unlike the P^ aeruginosa organisms, genotyping of the fecal streptococci 
did not produce any concise results. One hundred and ninety-two streptococcal 
isolates, 60 of which were frcm the sanitary sewer, were genotyped. A total of 
64 different REA patterns were identified among the isolates. 

Streptococcus faecal is subsp. faecal is (Table 12) was the only species 
that had the predominant genotypes 5 and 8 occurring in both the sanitary sewer 
and the priority storm sewer isolates. As stated previously Mundt (1973) has 
suggested that S^. faecal is isolates from human feces will produce an acid curd 
in litmus milk. Hcwever, no relationship between the 5 and 8 pattern isolates 
and those that produced an acid curd was observed. Although the S^ faecium 
isolates were distributed among 31 different REA patterns, genotype 9 was found 
in all three sites of the sanitary sewer and genotype 5 was recovered from all 
sites in the priority storm sewer. S^ faecium subsp. casseliflavus (Table 13) 



- 45 - 



s 



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(N^^O'^r^fn.Hn'^inrNiDr^co 



- 46 - 



c 



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- 47 - 
had 21 different REA patterns that were widely distributed among the isolates 
from different sources. In general, no relationship between the source 
of isolation and the genotype could be found among the 192 streptococcal 
isolates studied. 

More insi(^t into the origin of fecal wastes in storm sewer lines was 
provided by the speciation of fecal streptococci (Tables 59 to 63 , Appendix) . 
The results showed that S^ faecium tended to be equally represented in all 
sanple categories. On the other hand, S^ faecal is subsp. faecal is (Fig. 7) was 
found more frequently in sanitary and priority storm sewers than in surface 
runoff and non-priority sewers. In contrast, S^ faeccium subsp. casseliflavus 
(Fig. 8) predominated in non-priority storm s&/jer water and was notably evident 
in storm water runoff. The organism was virtually nonexistent in sanitary 
sewage. Levels of S^ faecium subs, casseliflavus in priority storm sewers were 
intermediate between the counts found in sanitary sewage and in the non- 
priority storm sewer. These results concur with our previous data (Sey fried, 
Harris, Young, 1986 uipublished) \A*iich showed that S^ faecium subsp. 
casseliflavus could be isolated from animal feces but not from human fecal 
specimens. 

Summary of 1988 Survey Results 

1. The levels of fecal coliforms, Escherichia coli . P^ aeruginosa and 
Bi f idobacter ium sp. suggest that there is an irrpact near site A in the 
storm sewer line that may be due to human feccil pollution. 

2. While Bi f idobacter ium breve and B^ adolescentis are found in human feces, 
they cannot be used to differentiate human from animal sources of 
pollution because they can be isolated from animals such as dogs and 
chickens. 



- 48 - 



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- 50 - 

3. Genotyping of Bifidobacterium isolates may provide a more precise method 
of source differentiation, but further work has to be done to find a 
suitable genotyping method. 

4. p^ aeruginosa genotyping may be of value in tracing sources of pollution. 
Serotyping, however does not provide results that are specific enough. 

5. Genotyping of fecal streptococci is not reccanmended as a method of source 
determination since the wide variety of patterns produced yield 
inconclusive results. 

6. Speciating fecal streptococci is a useful means of characterizing sewer or 
storm water content. For exaiiple, S^ faecal is subs, faecal is is found 
predominantly in sanitary and priority storm sewers whereas S^ faecium 
subsp. casseliflavus has not been isolated from human feces. 

DISCUSSICN OF ADOmCNAL FRUBCES 

A. Gcnparative Stixay of the Survival of Indicator Bacterial Species by Eric 
Bauer (see Appendix) 

Ihe conplete project is outline in the Appendix. The study had two 
objectives. One was to conpare the die-off rates of E^ coli , P^. aeruginosa , S^ 
faecal is var. faecal is . S^ faecium and B^ longum at room tenperature and at 
15° C. The second was to examine the lethality of chlorine on Ei coli . P^ 
aeruginosa . S. faecium var. casseliflavus and B^. breve . 

The results of the project showed that B^ longum died off more quickly 
than the other organisms at room tenperature. However, at 15° C, the usual 
tenperature in a storm sewer, P^ aeruginosa . E^. coli . and B^. longum all had a 
shorter life span than the streptococci. Thus these three organisms would be 
better indicators of recent pollution in storm sewers than fecal streptococci. 

Of the organisins tested, B^ breve appeared to be the most sensitive to 
chlorine treatment. The combined characteristics of sensitivity to adverse 



- 51 - 
conditions and ability to survive for only a relatively short period of time 
outside the body sujport the proposal that Bifidobacterium species would be 
useful irKlLcators of fresh fecal contamination. 

B. lliB Isolaticfi and Identificaticn of Bifidobacteria frcm Fecal and Sewage 
Samples by In-ja Huh (see Afpendix) 

An attempt was made in this study to find an optimal medium for the 
recovery of bifidobacteria from human and animal feces and from sewage. A 
ccffrparison was also made with the HBSA medium designed to isolate sorbitol- 
fermenting species of bifidobacteria. (In HBSA, lactose is replaced by 
sorbitol) . I 

Ihe results showed that the YN-17 medium yielded fewer false positive 
target colonies than HBSA (5% and 10%, respectively) • Ihe majority of false 
positive targets were found to be fecal streptococci. Lithium chloride, in 
concentrations of 0.3% and 0.4%, was added to YN-17 and HBSA to inhibit 
streptococcal grcwth; however, it was not successful in reducing the 
contaminant levels in these media. 

The YN-17 medium was developed by Mara and Oragui in 1983. It was found 
in this study that colonies the authors described as bifidobacteria (i.e. blue 
centre with pale green pjeriphery) were, in fact, a mixture of bifidobacteria 
and streptococci. The characterization profiles indicated that the smaller 
dark green/blue flatter colonies were bifidobacteria and those exhibiting a 
pale green periphery were contaminating streptococci or, more frequently, a 
mixture of the two organisms. Any colonies that were pale green in colour and 
did not contain a darker centre were found to be streptococci. 

Sorbitol-fermenting (B^. breve ) species were isolated from human and cat 
feces on the HBSA medium. It should be noted hcwever that none of the cat 
isolates were able to ferment sorbitol in subsequent tube tests. 



- 52 - 
Bi f idobacterium adolescentis was the most cxanmon species of bifidobacteria in 
sewage and human fecal samples. This organism was not found in cats. 

The data shewed that compared with E^ coli levels, bifidobacteria counts 
were 100 times greater in feces and 10 times greater in se^rage saitples. 

C. A Study of the Survival of Bifidobacteria and their Rale in Vfater Quality 
CCntrol by Sheila Shibata (see Appendix) . 

The main objectives of this research project were to determine the most 
suitable mediim for the isolation of bifidobacteria from feces and from 
environmental samples and to compare the in vitro survival characteristics of 
bifidobacteria and E^ coli . The results of the assessment of YN-17, MRS and 
MFN media showed that the MRS medium recovered not only Bifidobacterium sp. but 
high levels of lactobacilli and fecal streptococci as well. YN-17 and MEM were 
equally selective for bifidobacteria but YN-17 had an advantage in that it 
required a 24 h shorter incubation period. Bifidobacteria were found to exist 
in feces at a 1000-fold higher concentration than E^ coli . The sorbitol- 
fermenting bifidobacteria species, however, were present in feces in numbers 
conparable with E^ coli (i.e. 10^) . 

When bifidobacteria and E^ coli were added to dialysis diffusion chambers 
and suspended in Lake Ontario water it was found that Bifidobacterium sp. died 
off within 24 h of exposure vAiereas E^. coli died off more slowly and could 
still be isolated after 48 h or more. 

Since bifidobacteria are found in high levels in feces and have a tendency 
to die off rapidly in the environment these organisms shew promise as 
indicators of recent human fecal pollution. 



- 53 - 

D. Clostrldimn perfrinqens and Bifidobac±erii3m sp. as Trac3ers in Storm Sewers 
by Eric Hani (see /^peixiix) 

This project examined the feasibility of using C^ perfrinqens and 
Bifidobacterium sp. as indicators of fecal pollution from warm-blooded animals. 
The data shewed that both indicators were present in high concentrations in 
feces but that bifidobacteria could be isolated on a more consistent basis. 
The graphs indicated that bifidobacteria levels were hi(^ in both sanitary and 
hi^ priority storm sewage. Clostridium perfrinqens densities, on the other 
hand, were conparatively lew in the hi^ priority storm sewage. The histogram 
giving the geometric mean concentrations of E^ coli, C^ perfrinqens and 
bifidobacteria showed that levels of all three indicators tended to be hi(^er 
in the sanitary and high priority storm sewage sairples in conparison with the 
non-priority storm sewer sairples. Clostridium perfrinqens could not be 
recovered fran one non-priority storm sewer sanple. There did not appear to be 
any major trends with respect to source vhen the ratios of the three indicator 
orrganisms were coirpared (Table E-1) . 

In a random sanpling of human, cat and dog feces, bifidobacteria levels 
were found to be hi^er than E^ coli in humans. Although C^ perfrinqens was 
recovered frcm the cat fecal saitple, no bifidobacteria were found. As 
previously mentioned, Mara and Oragui (1983) found sorbitol fermenting species 
of Bifidobacterium to be exclusive to human feces. However, this reaction does 
not appear to be a practical criterion for source tracing since only a small 
percentage of the sanitary sewage isolates were sorbitol fermenters. 



- 54 - 

E. Chctracterizaticns of Pseudciiicr>as aerucrinosa fran Storm and Sanitary Sewers 
by Rita Harmandayan (see i^pendix) 

This project was designed to investigate the use of serotyping and 
genotyping to distinguish P^ aeruginosa strains fron human and non-human 
sources. The data shewed that levels of P^ aeruginosa were close to 1 x lO'* in 
sanitary sewage and 1 x lO-'- in storm sewage sanples. Approximately equal 
numbers of isolates fron both sources were serotyped using the Difco serotyping 
kit number 3081-32 ( Pseudomonas aeruginosa antigen kit). Serotype 0:6 was the 
most frequently indentified serotype in both the storm and sanitary sewers. 
Serotypes 0:1 and 0:11 were also commonly isolated from both sources. Serotype. 
0:16 was recovered from storm sewers only, with a frequency of 5.4%. Serotype 
0:10, on the other hand, was isolated solely from sanitary sewers with a 
frequency of 21.9%. It is possible, therefore, that the presence of type 0:10 
may be indicative of human fecal contamination. 

To determine if identical genotype patterns were present within each 
serogroup, restriction enzyme analysis was performed on isolates with common 
serotypes. Of the six different restriction enzymes used to cut the genomic 
K^A, Sma I produced the best banding pattern on polyacrylamide gels. The 
results shewed that not all isolates with the same serotype possessed the same 
genome pattern. Ihis is e>plained by the fact that the genes responsible for 
serotyping the 0-Ag comprise a small percentage of the total genome and that 
other genes besides those that code for the 0-Ag will appear v*ien the total 
chromosomal CNA is cesrpared. Ihus, the REA technique was shown in this study 
to be highly specific; it further subdivides one serotype into different 
genotypes. In future, a probe could be developed from either total chromosomal 
OIA or a specific fragment common to human strains that would allow 
hybridization against other P^ aeruginosa strains. The probe would help to 
differentiate between isolates from human and non-human fecal waste. 



- 55 - 

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• - 56 - 

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- 57 - 

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■ - 58 - 

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Infec. Dis. 130 (suppl.): S47-S52. 



- 60 - 

APPENDIX A 
Tsitle 14 

Sanpling Dates far Dry and Wet Weather Surveys 



Year 



Date 



category 



1986 

1987 

1988 



October 21, 22 and 28, November 18 

June 10, 11, 12 and 19 
August 17 

June 20, 21, 28 
August 15, 22 



Dry Weather I 

Dry Weather II 
Dry Weather 

Dry Weather 
Dry Weather 



1987 



July 14 
September 18 
October 21 



Wet Weather I 
Wet Weather 
Wet Weather 



- 61 - 



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- 116 - 



AFraNDIX B 

CCMPARATIVE STUDY OF THE SURVIVAL 
OF INDICATOR BACTERIAL SPECIES 

Eric Bauer, Department of 
Microbiology, University of Toronto 



- 117 - 
TABLE OF OCWTEyiS 

Page No. 

I. INTRDDUCnON 121 

II. OBJECnVES OF RESEARCH 126 

III. MATERIALS AND METHODS 127 

Bacterial cultures 127 

Growth cycle determination 127 

Membrane filtration techniques for the isolation 129 
of test organisms 

Enumeration of colony forming units per mL of 130 
test bacterial cultures 

Chlorine test procedure 131 

IV. RESULTS 

Growth cycle determination 134 

Bactericidal effect of chlorine 138 

V. DISCUSSION 155 

VI. RECCMIENDATIONS • 162 

VII. APPENDIX 163 

VIII. REFERENCES 169 



• - 118 - 
UST OF T ARTRR 
Table 



Page 



B-1 Media and Incubation Parameters for Enumeration 128 
of Selected Bacterial Groi-ps 

B-2 Colony Forming Units per Millilitre at Rocan 135 
Terrperature 

B-3 Colony Forming Units per Millilitre at 15 136 

degrees Celsius , • , 

B-4 Effect of Chlorine at a pH of 6.0 ^ ' 150 

B-5 Effect of Chlorine at a pH of 9.0 150 

B-6 Effect of pH on Forms of Chlorine 160 



- 119 - 
UST OF FIGURES 

Figure Page 

B-1 Colony Forming Units per mL vs. Time at Room 139 

Temperature 

B-2 Colony Forming Units per mL vs. Time at 15 140 

degrees Celsius 

B-3 E. coli CFU/mL vs. Time at Room Tenperature 141 

and 15 degrees Celsius 

B-4 P. aeruginosa CFU/mL vs. Time at Room 142 

Temperature and 15 degrees Celsius 

B-5 Strep , on m-Ent CFU/mL vs. Time at Room 143 

Tenperature and 15 degrees Celsius 

B-6 Strep , on m-E CFU/mL vs. Time at Room 144 

Temperature and 15 degrees Celsius 

B-7 B. longum CFU/mL vs. Time at Room Terrperature 145 
and 15 degrees Celsius 

B-8 Magnification of Colony Forming Units per mL 146 

vs. Time at Room Tenperature 

B-9 Magnification of Colony Forming Units per mL 147 

vs. Time at 15 degrees Celsius 

B-10 Magnification of E. coli CFU/mL vs. Time at 148 

Room Tenperature and 15 degrees Celsius 

B-11 Magnification of P. aeruginosa CFU/mL vs. Time 148 
at Room Temperature and 15 degrees Celsius 

B-12 Effect of 0.2 ppm Chlorine on Various Organisms 151 
at a pH of 6.0 

B-13 Effect of 0.4 ppm Chlorine on Various Organisms 152 
at a pH of 6.0 

B-14 Effect of 0.2 ppm Chlorine on Various Organisms 153 
at a pH of 9.0 

B-15 Effect of 0.4 ppm Chlorine on Various Organisms 154 
at a pH of 9.0 



- 120 - 
UST OF ABEREVIATrONS 

Organisms : 

E. coli - Escheric±iia coli 

B. breve - Bifidobacterium breve 

B. lonqum - Bi f idobacter ium longum 

P. aeruginosa - Pseudomonas aeruginosa 

S. faecal is var, faecal is - Streptococcus faecal is 

variety faecal is 

S. faecium var. casseliflavus - Streptococcus faecium 

variety casseliflavus 

CFU - Colony forming unit 

°C - Degrees celsiiis 

dH20 - Distilled water 

E - Exponential 

pH - Hydrogen ion concentration 

mL - millilitre 

ppm - Parts per million 

PSI - Pounds per square inch 

vs. - versus 

M - nxDlar 



- 121 - 

nraojJCTicN 

Fundamental to the interpretation of data concerning surface 
uater quality is an understanding of the growth cycles and viability 
of fecal indicator bacteria and enteric pathogens disseminated by 
waterways. In the recent past public awareness pertaining to the 
contamination of surface waters has resulted in increasing efforts 
to control this type of pollution. The potential health hazard, in 
v/hich waterways may act as both a vehicle and a reservoir for 
agents of infectious disease, is apparent. The prevention of. 
epidemics caused by such organisms as Salmonella typhi . Salmonella 
typhimurium . Vibrio cholerae . Shigella species, Giardia lamblia , 
enterotoxigenic E. coli and P. aeruginosa may only take place with 
the adequate control of fecal contamination (Gyles, 1984) . 

The main concern of this study deals with the comparative 
analysis of the life cycle of E. coli , P. aeruginosa , S. faecal is 
var. faecal is , S. faecium and B. longum . The theoretical aspect 
of this investigation is concerned with obtaining an organism of 
short life duration so that it may be used as an indicator of 
recent fecal contamination. Although the detection of fresh fecal 
contaminants within any surface water body is paramount, the 
impetus for this research came from the ever growing problem of 
storm water sewage contamination. It is for this reason that 
growth curves for the indicator organisms were established at both 
room tenperature and 15°C (approximate sewer tenperature) . 



■ - 122 - 
The original design strategy behind storm water sewage 
channels was the alleviation of excessive runoff - water from 
urbanized areas. Theoretically these storm sewers should have no 
human fecal input, hcwever, as reported by the Toronto Area 
Watershed Management (TAWM) , this is not the case. Fecal 
pollution within the Humber and Don Rivers does result in storm 
water sewage serving as a contributor. 

The organisms selected for this study were chosen for the 
following reasons: 

E. coli ' ' 

This organism is constantly found in the human intestine 

in large numbers. 

The fate of the coliform bacteria reasonably reflects 

that of the pathogenic bacteria, althou^ the life 

e>pectancy of the fecal coliforms is normally longer 

than that of intestinal pathogens. 

This organism is easy to isolate and enumerate in the 

laboratory environment and is normally not pathogenic 

(Schuettpelz, 1969). 

S. faecal is var. faecal is / S. faecium 

A hi^ percentage of the strains have properties which 

enable their source to be identified with considerable 

certainty. 

Members of the fecal streptococci group are often used 

to signify the presence of intestinal pathogens 

(McFeters et al., 1974). 



- 123 - 
P. aeruginosa 

Ringen and Drake (1952) have shewn that this organism 
was not isolated from locations free of human habitation 
or waste material. 

This opportunistic pathogen is particularly associated 
with the hi^ incidence of otitis externa in surface 
water related areas (Levin and Cabelli, 1972) . 

B. lonqum 

The strictly anaerobic requirement of this organism 

makes its inclusion in a study of growth within a sewer 

environment of interest. 

A relatively small amount of work has been done on this 

organism and its membership in the group of fecal 

organisms. 

The method of choice for the enumeration of the indicator 
organisms was that of membrane filtration. This technique was 
selected because of the relative sinplicity of the procedure, the 
capability of obtaining results within 24 hours, instead of the 48 
to 96 hours demanded by the Most Probable Number test and 
finally, the larger volume of samples v*iich may be analyzed ma3djTg 
the obtained results more representative. 

The second portion of this survival research project 
concerns iself with the effect of chlorination on the following 
organisms: E. coli . P. aeruginosa . S. faecium var. casseliflavus 
and B. breve . The lethality of uncombined chlorine, in the form 
of unionized hypochlorous acid has made this chemical the most 



- 124 - 
widely used reagent for the disinfection of water distribution 
systems and reservoirs (Ridgway and Olson, 1982) . Of more recent 
interest, hcwever, has been the possible application of the method 
of chlorination for purposes of sterilization. Factors such as 
concentration of the chlorine used, the pH of the enviromnent in 
vv*iich the chlorine is dispensed and the free chlorine residual 
must all be considered vdien analysing the effectiveness of this 
agent as a bactericide (Seyfried and Fraser, 1979) . 

The action of chlorine, from the chemical point of view, may 
be summarized as follows: ' 

1. Hydrolysis of chlorine: 

CI2 + HOH =- HOCl + ir^ + CI" 

Above pH values of 5.0, the hypochlorous acid 
(HOCl) dissociates in aqueous solution and forms an 
equilibrium with hydrogen and hypochlorite ions, 
with the relative amount of each species dependent 
upon the pH. 

2. Dissociation of hypochlorous acid: 
HOCl = H^ + OCl" 

The bactericidal activity is a result of the 
hypochlorous acid \«*dch is free chlorine, while the 
hypochlorite ion has limited killing ability, but 
acts as a reservoir of available chlorine. 

3. Combined or "stabilized" chlorine: 



- 125 - 
Ihis is the reaction of chlorine with such 
materials as ammonia or its salts, cyanuric acid, 
sulfamic acid or urea. Chlorine in this form is 
virtually ineffective, having a killing time of 
approximately one thirtieth that of hypochlorous 
acid (Black et al., 1970). 

The final point of interest concerning the action of 
chlorine is related to its biochemical action upon the bacterial 
cell. It is at this point that differences in lethality exist, 
when environmental conditions are kept constant between organisms. 
Recent proposals, by Canper and McFeters (1979) as to the mode of 
action of chlorine on the bacterial cell include: 

unbalanced metabolism after the destruction of key 
enzymes 

disruption of protein synthesis 

oxidative decarboxylation of amino acids 

reactions with nucleic acids, purines and pyrimidines 

formation of chloro derivatives of cytosine 

creation of chromosomal abberations 

induction of deoxyribonucleic acid lesions with the 
acconpanying loss of deoxyribonucleic acid transforming 
ability 

inhibition of oxygen uptake and oxidative 
phosphorylation coupled with the leakage of some 
macromolecules 



- 126 - 
OBJECTIVE OF RESEARCH 

The objectives of this research project were to: 

1. Study the growth cycle of E. coli . P. aeruginosa , £5. 
faecal is var. faecal is , S. faecium and B. longum . The 
results of this corrparative analysis may then be applied to 
the development of a methodology for detecting the source of 
sanitary waste pollution in surface water bodies, since the 
presence of a short life expectancy organism will be 
indicative of recent pollution. 

2. Of growing interest is the use of chlorine, not only as a 
disinfectant, but also as a potential sterilant. 
Manipulation of the concentration of chlorine, as well as 
the hydrogen ion concentration of the environmental setting, 
will provide insight into the effective lethality of chlorine 
on E. coli , P. aeruginosa , S. faecium var. casselif lavus . and 
B. breve. 



- 127 - 

MATERIALS AND METHODS 

3.1 Bacterial cultures: 

Seven bacterial cultures were used in this study. 

Four laboratory strains v^iich included E. coli , P. 

aeruginosa , S. faecal is var. faecal is , and S. faecium . 

Two environmental isolates which included B. longum and 

S. faecium var. casseliflavus . 

One fecal saitple isolate, B. breve , taken from a 

chicken. 

The environmental isolates and the fecal sample isolate were 
positively identified using biochemical testing procedures, as 
outlined in Bergey's Manual of Determinative Microbiology (1985) . 

3.2 Growth cycle determination: 

E. coli , P. aeruginosa , S. faecal is var. faecal is and S. 

faecium were all grcwn for 24 hrs. , aercbically, in lOmL 

of nutrient broth, at 35°C. 

B. lonqum was grown for 48 hrs., anaerobically, in lOmL 

of MRS broth at 35°C. 

Two 250mL culture flasks were autoclaved for 15 minutes 

at 121°C / 15 PSI. To these flasks, 90mL of filter 

sterilized pond water was then aseptically added. 

To ensure that sterility was achieved, spread plates of 

the sterilized water were made on nutrient agar. 

Once these culture flasks were prepared, the five 

cultures 



- 128 - 



Table B-1 



Media and Incubaticn I^rameters for 
Qiimpratlcn of Selected Bact-prial Grtx^s 



BACTERIAL 
0«XJP 



ZNCUBAT- TIME 
MEDIUM* TEMP 'C (HRS) 



TARGET Cr)I£NY ICREHDLDGY 



E. Coli 



m-TEC 44.5 23+1 - flat greenish/yellow colonies 

- circular 



NA 



35 



24 - small beige colonies 
- circular 



P. aeruginosa 



m-PA 41.5 



48 



convex brownish-green or tan 
circular 



NA 



35 



24 



spread out and of irregular 
form 



S. faecal is 
var. faecal is 



m-EOT 



35 



48 



maroon, red, or pink colonies 
circular and raised 



S . f aecium 



m-E 



41.5 



48 



blue/pink colonies 
pulvinate to umbinate 



S^ f aecium BHI 

var. casseliflavus 



35 



24 



very small yellow colonies 
convex to pulvinate 



B. loncami 



YN17 
Blue 



35 



48 



smooth to undulating surface 
convex to pulvinate 
soft, moist, slimy blue 
colonies 



B. breve 



MRS 



35 



24 



convex to pulvinate 
ivory coloured 
muccoid and soft 



- 129 - 
were each diluted in lOmL of their respective broths and then aseptically added to 
the culture flasks. 

These dilutions were pr^)ared so as to start with an initial colony 
forming units per mL of approximately 10E5 to 10E6. 

The bacterial culture dilultions were made in duplicate, as were the 
culture flasJcs, in order that the growth cycles could be examined at both 
15°C and room tenperature. 

Both the tenperature of the 15°C incubator, as well as the tenperature of 
the laboratory were recorded daily: 
Room tenperature range - 23°C +/~ 2°C 

15°C incubator - 15°C +/" 2°C, with the exception of Day 9 

of the study when the incubator tenperature 
fell to 8°C. 

3.3 Membrane filtration technique for the isolation of the test organisms. 

The procedure followed for the membrane filtration was that as outlined in 
(Standard Methods for the Examination of Water and Waste Water, 1971) . 
Sanples were removed from both the 15°C and the room tenperature culture 
flasks over a 40 day experimental period. 

The first determination (Day 0) was taken immediately following the 
initial incubation of the bacterial cultures within the pond water. 
- Sanple filtration, for the purpose of enumeration, was always carried out 
between 9:00AM and 12:00FM and the determination dates were recorded. 
Sanples were first diluted in 99mL phosphate buffer dilution blanks (see 
i^pendix) and then filtered (Gelman filters) . 
^^proximately lOOmL of phosphate buffer wash water (see Appendix) were 



- 130 - 
divided into three aliquots for consecutive rinsing of the saitple and 
membrane filtration apparatus. 

- After washing the membrane filters were placed on the growth medium, 
taking care to ensure that no air bubbles were trapped under the filter. 
The growth medium, incubation parameters and target morphology for all of 
the test organisms may be found in Table 1. 

3.4 Enumeration of colony forming units per mL of test bacterial cultures: 

- For all organisms, with the exception of B. lonoum , target colony 
morphology was specific to the isolating growth medium. 

The ability of fecal streptococci to grow on the YN17 Blue 
( Bifidobacterium isolating) medium made morphological examination of 
typical colonies from this medium essential. 

- Typical colonies were selected frcm the membrane filter, from the YN17 
Blue medium and microscope slide preparations were made. 

- Bi f idobacter ium characteristic morphology; long curved, club-shaped, 
swollen or dumb-bell shaped rods, vs*iich may also be bifurcated. 

- At the point in time when this type of cell morphology was no longer seen, 
typical colonies were selected and exposed to biochemical testing 
(Bergey's Manual of Ceterminative Microbiology, 1985). 

Ihe two fecal streptococci, S. faecal is var. faecal is and S. faecium , were 
both included into the study so that it could be determined which had a 
shorter life span. Typical colonies were selected from the m-E medium 
(see Appendix) and biochemically tested so as to permit the calculation of 
a ratio of surviving numbers of the two fecal streptococci. 

- The fecal streptococci were also grcwn on both m-Ent and m-E (see 



- 131 - 
Appendix) so as to cxmpare the consistency of counts between the colony formir.-- 
units on the two different media. 

The appearance of the colonies on m-E agar was variable, and biochemical 
tests were perfornved on two different types of colonies in order to detenr.ir.o 
whether these differences represented the two different fecal streptococci . 

3.5 Chlorine test procedure: 

The bacterial effect of chlorine on the test organisms was determin&.l 

using the experimental procedure of Seyfried and Fraser (1980) . 

All glassware which came into contact with the experimental chlorir.-:- 

concentrations was acid-washed and then treated overnight with a calcicc. 

hypochlorite solution of concentration O.Olg per lOOOmL. 

A stock chlorine solution of 0.5g calcium hypochlorite to 500mL of dii.L 

was prepared. A sterile sodium thiosulfate solution (0.35g / 500mL) -..a?- 

also prepared and was used to neutralize the chlorine. 

Reaction tubes were filled with 8mL of dH20 and then autoclaved for i-I 

minutes at 121°C / 15 PSI. With sterilized IM sodium hydroxide and : 

hydrochloric acid the pH of the reaction vessels was then adjusted to 

and 6.0, respectively. 

E. coli. P. aeruginosa and S. faecium var. casseliflavus were all grov.Ti 

for 24 hours, aerobically, in lOmL of nutrient broth at 35°C. 

B. breve was grown anaerobically for 24 hours in lOmL of MRS broth at 35°C. 

Two variables were incorporated into the experimentation of the 



- 132 - 
bactericidal effect of chlorine on the test organisms, through the follov.-i:-.o 
experimental trials: 

(A) Effect of chlorine at a concentration of 0.2 ppm and a pH of 6.0 

(B) Effect of chlorine at a concentration of 0.2 ppm and a pH of 9.0 

(C) Effect of chlorine at a concentration of 0.4 ppm and a pH of 6.0 

(D) Effect of chlorine at a concentration of 0.4 ppm and a pH of 9.0 
Bacterial cultures were centrifuged and then washed twice with 5mL aliqucts 
of sterile dH20. The washed cultures were then resuspended in 5mL c: 
sterile dH20 and thoroughly mixed. 

The estimation of the chlorine concentration was done before each experimenCLi. 

trial and made use of the diethyl-p-phenylenediamine (DPD) test (Americar. 

Public Health Association, 1971) . 

At this point l.OmL of culture was removed and diluted in 99mL of phospharc- 

buffer dilution blank. This suspension was then placed on ice for lat^-r 

further dilution and subsequent membrane filtration. In this manner •::._ 

initial concentration of bacteria per mL could be calculated. 

The test system consisted of 8mL of sterile pH adjusted dH20, ImL of test 

culture suspension and ImL of chlorine. 

The cells were exposed to the particular chlorine concentration for i 

minute. After this contact period ImL of the neutralizing solution of : 

sodium thiosulfate was added to stop the reaction. 

ImL of suspension was then removed from the reaction tube and placea ... 

the 99mL phosphate buffer dilution blanks. 

Membrane filtration was performed first on the chlorine exposed cells ar.L; 

then on the initial cells which were on ice until this point. Each teiic 



- 133 - 

organism was filtered individually, at the "before and after" e>posures, 

so as to reduce the possibility of any cross contamination. 

Referring to Table 1, the grcwth media, incubation parameters and typical 

colony morphology may be seen (Note: In this case selective media was not 

used. Instead, less stressful media was chosen; nutrient agar, BHI agar 

and MRS agar) . 

Ihe experimental procedure was performed at a teitperature of 23 +/~ 2°C. 



• - 134 - 

RESULTS 
4.1 Growth cycle determination: 

Tables 2 and 3 shew the growth of E. coli, P. aeruginosa . S. faecal is var. 
faecal is / S, faecium on m-Ent and m-E media, and B. lonqum . Enumeration of test 
bacterial cultures was performed at room tenperature and 15°C (Table 2 and Table 3, 
respectively) . 

As can be seen in the data of the rocm tenperature detennination, the fecal 
streptococci attained the highest numbers of colony forming units per mL. Although 
all organisms showed a period of substantial increase in growth, this period was 
extremely short for B. lonqum , lasting for only the first day. In conjunction with 
this, B. loncpjm was the first organism to die off; as well, P. aeruginosa was the 
only other organism to die before the end of the 40 day experimentation period. 
Some fluctuation in growth was recorded, namely the data collected does not reflect 
a steady increase in growth follcwed by a decline period. Examination of the 
results of the fecal streptococci illxostrate a high degree of consistency between 
the colony forming unit per mL values on m-Ent with that of m-E. 

At the 15°C determination, the most striking feature is the rapid "die-off" of 
E. coli , P. aeruginosa and B. longum . These three organisms all perished sometime 
between Day 8 and Day 13. As with the rocm tenperature determination, the fecal 
streptococci again were most abundant; however, the numbers obtained at 15°C were 
not quite as high as those at room tenperature. Another notable occurrence is that, 



- 135 - 
Table B-2 
Colony Forming Units per Mlllllitre at Room Temperature 



TIME 


E. coll 

aaaaasaaaaasa 


P. aeruginosa 


Strep, 
on Ment 

isaaaaaaaaaaa: 


Strep, 
on ME 

ssaaaaaaassssa: 


i.' 

ssaas 


, lonqum 


(days) 




saaaaaaa: 





1.80OB+05 


2.695E-t-05 


1.320E-f06 


1.815E+06 


5. 


.840E-I-05 


1 


2.330B+07 


1.090E^06 


3.980E+06 


3.730Ef06 


7. 


.610E+05 


4 


3.750E+07 


1.875E+06 


4.390E+07 


5.540E+07 


5, 


,370E+05 


5 


3.070E+07 


3.480Ef06 


1.223E+08 


1.383E+-08 


5, 


.025E+05 


6 


1.925E+07 


1.800E-I-06 


1.335E+08 


1.250E+O8 


1, 


.395E+05 


7 


4.900E+07 


1.123Ef06 


6.333B+07 


1.405B+08 


2. 


.230E+05 


11 


4.120E+07 


2.880E+07 


5.750E+07 


1.363E4-08 


9, 


.720E+04 


13 


3.950E+07 


4.0O0E+07 


2.330B+09 


6.150E+07 


0. 


.0OOE4-OO 


15 


4.500B+07 


7.850E-t-08 


3.790E+08 


1.920E+09 


0. 


.OOOE+00 


19 


2.100E+07 


8.640B+08 


3.180E-I-08 


5.960E+08 


0, 


.OOOE+00 


21 


1.840E-«-07 


3.500E+07 


2.880E+07 


1.660E-»-08 


0. 


.OOOE+00 


25 


1.760E+07 


4.740E^06 


2.610B+07 


3.910E+07 


0. 


,0OOE+OO 


27 


1.400E+07 


6.320E+05 


2.800E+07 


4.500E+07 


0, 


,OO0E+OO 


29 


9.200E+06 


4.640E4-04 


2.700E+07 


3.900E+07 


0, 


.OOOE+00 


36 


6.800E-t-06 


1.300E+02 


2.400E+07 


3.000E+07 


0. 


.OOOE+00 


40 


8.340E+05 


0.000B4-00 


1.240E+04 


1.840E+07 


0, 


, OOOE + 00 



- 136 - 

Table B-3 
Colony Forming Units per Mlllllltre at 15 degrees Celsius 



TIME 


E. coll 


P. aeruqlnosa 


Strep. 


Strep. 


!-• 


. lonqum 


(days) 






on Ment 


on ME 






asasass 


asssaaassasa: 


saaaaaaasaaaaaass 


:aasasaaaaaaa 


aaaaaaaaaaaaaaaa 


aaaaaasaaa: 





2.020E+05 


3.170B+05 


1.350B+06 


1.625E-I-06 


4 • 


,900E-t-05 


1 


1.420E-«-05 


1.903E-^05 


1.453E-t-06 


1.500E-f06 


4 . 


.450E-t-05 


4 


1.260B+05 


2.760E-»-05 


4.640E^06 


4.685E+06 


4 


.210E+05 


5 


8.850E+04 


1.085E-t-05 


9.850E-t-06 


1.005E-I-07 


1 • 


.873E-h05 


6 


2.020B+05 


3.245E4-05 


4.800E-t-07 


3.070Ef07 


9 


.730E+04 


7 


2.087E+06 


3.200E-«-04 


3.270E-t-08 


2.880E-t-08 


8 


.060E-I-04 


11 


4.100E-I-04 


O.OOOE-t-00 


1.900E-t-08 


2.437Ef08 


2 


.120E+04 


13 


2.000E+02 


O.OOOE-t-00 


2.720E-t-08 


2.725E-t-08 


0, 


.OOOE-i-00 


15 


O.OOOB+00 


O.OOOB+00 


2.310E+08 


2.040B>08 


0. 


.000E4-00 


19 


O.OOOE+00 


O.OOOE-t-00 


1.130E-t-08 


1.090E-I-07 


0, 


.OOOE-l-00 


21 


O.OOOE+00 


O.OOOE+00 


7.650E-I-06 


9.130B-I-06 


0, 


,000E+00 


25 


O.OOOE-l-00 


O.OOOE-t-00 


1.190E-t-04 


1.850E-I-04 


0, 


.OOOE-l-00 


27 


O.OOOE+00 


O.OOOE-t-00 


2.400E^04 


2.200E-I-04 


0. 


,000E+00 


29 


O.OOOE-t-00 


O.OOOE-i-00 


1.900E-I-04 


2.500E-t-04 


0. 


.OOOE-fOO 


36 


O.OOOE^OO 


0.000E4-00 


1.400E^04 


2.800E<-04 


0. 


.OOOE+00 


40 


O.OOOE-t-00 


O.OOOE-t-00 


1.240E-t-04 


2.330E-I-04 


0. 


,OOOE-l-00 



- 137 - 
with the exception of the fecal streptococci on m-Ent, all other cases showed a 
decline immediately after incubation was initiated. Again, a degree of fluctuation 
within the growth cycle of the organisms was observed upon enumeration. 

In the case of B. longum . at both room temperature and 15°C, microscopic slide 
preparations of colonies from the Day 11 determination did possess cells of 
characteristic Bi f idobacterium morphology. On the Day 13 enijmeration, however, 
morphological analysis proved negative for Bifidobacteriiim and for this reason four 
typical colonies were selected from a YN17 plate containing 5 colonies, for 
biochemical testing (Bergey's Manual of Determinative Microbiology, 1985). The 
results obtained were all negative for Bi f idobacterium . but positive for 
Streptococcus . Due to the absence of positively identifiable Bifidobacterium all 
colonies v\^ch did grew were not enumerated and it was assumed that B. longum no 
longer existed. 

Figures 1 through 10 represent the graphical analysis of the results recorded 
in Tables 1 and 2. The following is an explanation of v*iat the respective Figures 
depict : 

Figures 1 and 2 are comparative graphs of the test organisms at both room 
temperature and 15°C. (Note: These Figures, as with all others in v*iich the fecal 
streptococci are found, do not distinguisli between S. faecal is var. faecal is and S. 
faecium but instead conpare the combined fecal streptococci as to their growth on 
either m-Ent or m-E growth media) . As can be seen on these graphs, the values for 
the fecal streptococci are enormous in conparison to the other organisms. For this 
reason, magnifications of these grap^is were required and may be found in Figures 8 
and 9. The fluctuations in growth are more readily appcirent when viewing the 
graphs, but in an attempt to compensate for those days on v*iich determinations were 
not taken, the "best-fit" line was drawn through the points. Two final points 



- 138 - 
worthy of mention include the rapid "die-off" of all onganisms but the fecal 
streptococci, at 15°C. Secondly, the fact that only E. coli demonstrates a period 
of growth followed by a short period of "die-off" and then another resurgence, at 
both tenperature settings. 

Figures 3 through 7 plot the results obtained at room temperature against those 
of 15°C, for each individual organism. Ihis permits the viewing and conparison of 
the tenperature settings, on the grcwth of the test organisms, without the added 
confusion of the results from the other organisms. In all cases the numbers of 
colony forming units per mL are drastically reduced at 15°C. This difference was so 
marked with respect to E. coli and P. aeruginosa that magnifications of their 
results were produced and may be found in Figures 10 aind 11. The fecal 
streptococci and B. lonqum seemed to suffer less from the reduced tenperature of 
15°C, rarely having CFU per mL enumerations which differed by even an order of 
magnitude. 
4.2 Bactericidal effects of chlorine: 

Tables 4 and 5 contain the results of chlorine exposure upon the test organisms 
E. coli . P. aeruginosa . S. faecium var. casseliflavus and B. breve . For all 
experimental situations, the exposure time to chlorine was for a 1 minute contact 
period. Table 4 is a record of the results obtained v*ien the pH of the reaction 
flask was 6.0, vtiile Table 5 illustrates the results obtained at ftfi 9.0. 

The fact that the bactericidal effect of chlorine is not only exposure and 
concentration dependent becomes quickly apparent when the percentage of organisms 
killed is conpared between the different pH settings. Also, the higher concentration 
of chlorine is much more lethal, at both pH. settings, than is the 0.2 ppm 
concentration. 



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- 149 - 
One ofcservation of particular interst is the tendency towards more effective 
killing, by chlorine, when it is functioning within an acidic environment. Only E. 
coli and S. faecium var. casseliflavus had a Icwer killing percentage (at 0.4 ppm) 
at the Icwer pH of 6.0. All other experimental determinations shewed more effective 
killing at pH 6.0. Ihis observation was particularly apparent in the killing of P. 
aeruginosa . ' ^ ^ 

Figures 12 tlirou^ 15 graphically d^ict analysis of the results of the 
chlorine experimentation. It is obvious that chlorine has lethal effects, from the 
negative slope of all the graphs, but more inportant is the drastic effect seen at 
0.4 ppm in coirparison to that of 0.2 ppm. At the two ends of the percentage killed 
spectrum lie E. coli . with the smallest percentage killed (0.2 ppm at pH at 9.0) and 
B. breve , experiencing the highest percentage killed (0.4 ppm at pH 6.0) . 



- 150 - 
Table B-4 

Bffect of Chlorine at a pH of 6.0 



ORGANISM INITIAL CFU/nL 



E. coll 

^. aeruginosa 

3 . faeclun 



1.99B+09 
2.38E+09 
1.69B4-09 



var . cassellf lavus 

B. breve 2.67E+09 



AFTER CHLORINE ADDITION 
. 2 ppa . 4 ppa 



3.1584-08 
1.77E+08 
1.52B4-08 

2.82E-f08 



1.31B-t-08 
8.25E+07 
8.65B4-07 

5.40E+07 



\ KILLED 
. 2 ppn . 4 ppm 



84.2 93.4 

92.6 96.5 

91 94.9 



89.4 



98 



Table B-5 



Bffect of Chlorine at a pH of 9.0 



ORGANISM 

E. coll 

£^. aeruginosa 



^. faecltim 
ar. cassellf lavus 



INITIAL CPU/mL 



AFTER CHLORINE ADDITION 
. 2 ppm . 4 ppm 



3.80E-»^09 
1.82B4-09 
2.80E-I-09 



1.08E-I-09 
3.47B4-08 
2.73E+08 



2.30E-t-08 
1.90B4-08 
1.20B-(-08 



\ KILLED 
0.2 ppm . 4 ppm 

71.6 93.9 
80.9 89.6 
90.3 95.7 



B- breve 



6.15S-I-09 



1.04E4-09 



3.16B-»-08 



83.1 



94.9 



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DlSOJSSICa^ 

The analysis of the growth cycles of E. coli, P. aeruginosa , S. faecal is var. 
faecal is , S. faecium and B. loncrum had as its goal the development of a methodology 
for the purpose of locating a bacterial indicator vrtiich could be used for the 
detection of recent fecal contamination within surface water bodies. This is a 
problem of growing public concern, since the potential health hazard such pollution 
creates is of grave inportance. The experimental trial at room teitperature served 
to act as a standard or means of ccmparison for those results obtained at 15°C. 
This teitperature was selected to simulate, on the basis of tenperature alone, the 
environment of a storm water sewer (Seyfried personal communication, 1988) . It has 
been reported by the Toronto Area Watershed Management (TAWM) that a major 
contributor to fecal contamination within the Humber and Don Rivers is the effluent 
from Toronto storm water sewers. 

The controversy over v*iich bacterial indicator to use, for the purpose of water 
pollution control policies, has resulted in much debate. Taylor et al. (1973), 
state that the enumeration of fecal coliforms provides a more precise measure of the 
potential health hazard in surface water bodies, than does the more omniscient total 
coliforms. According to Doran and Linn (1979) fecal coliforms are reported to be 
the most reliable indicator of fecal pollution of water; however, with these 
organisms the source of contamination may not be identified. To compensate for this 
the use of fecal streptococci has been suggested in order to differentiate fecal 
contaminants from human or other animal sources (Geldrich and Kenner, 1969) . 
Schuettpelz (1969) reports that enterococci approach numbers of total coliforms in 
sewage, but have the added advantage of not multiplying in water. 

It would appear that the trend to employ fecal streptococci as indicators of 
fecal pollution is growing, but problems with their acceptance still exist. 



- 156 - 
Firstly, fecal streptococci are present in smaller numbers in feces, sewage and 
polluted waters, and the easier quantification of fecal coliforms is inhibitory to 
their use. Ihe second major problem lies within the confusion which exists 
concerning the identity of fecal str^Jtococci, in particular, that of their 
ecological distribution (Levin et al. 1975) . To overcame this handicap a barrage of 
research has been performed in the field of developing media selective for specific 
fecal streptococci, so that their identity may be positively confirmed. Work done 
by Isenberg et al. (1970), Wie-Shing Lee (1972), Switzer and Evans (1974) and 
Brodsky and Schiemann (1976) is illustrative of only a minor saitpling of involved 
participants. Another attempt at overccining the barrier of being unable to 
identify the source of the fecal contamination has been to use the fecal coliform 
(FC) to fecal str^atococci (FS) ratio. Doran and Linn (1979) claim that an FC/FS 
ratio of greater than four is usually indicative of domestic waste water pollution, 
v*iile an FC/FS ratio of less than 0.7 may be associated with non-human animal 
wastes. 

Recently, P. aeruginosa has been considered as a potential indicator of fecal 
contamination from human sources. Wheater and co-workers (1978) have shown that P. 
aeruginosa was not found in a variety of animal feces and also that the presence of 
this organism in animals and soil was due to chance and the close proximity to man 
(approximately 15% of the human population contain P. aeruginosa as a norrral 
commensal microbe) , For this reason and because of the opportunistic pathogenic 
nature of P. aeruginosa it has been given consideration as an indicator of fecal 
contamination in surface water bodies. 

The final organism, B. longum , which was included in this growth study 
comparison is a member of a genus upon which a great deal of research has not been 
done. Classification and identification of Bifidobacterium species monopolizes the 



- 157 - 
research of this organism. Its membership as an intestinal bacteria and 
siibsequently as a fecal organism protpted its inclusion. 

Concerning the 15°C temperature setting, experimental results shewed that P. 
aeruginosa had the shortest life span. As well, E. coli (fecal coliform) and B. 
lonqum have life expectancies only slightly longer than that of P. aeruginosa . The 
two fecal streptococci survived longest, and were still graving when experimentation 
was terminated. The problem encountered here was that the continued growth of both 
S. faecal is var. faecal is and S. faecium prevented the determination of which 
oinganism died first. Biochemical testing done at Day 30 on the fecal streptococci 
resulted in the identification of both species, with continued "picking" being 
unfeasible because of time limitations. 

Despite the stated goal of this reasearch, namely the determination of a 
bacterial indicator of recent fecal contamination of surface water bodies, the 
theoreticcil approach was to simulate an environment like that of a storm sewer, 
since these conduits act as a major vehicle for the transport of fecal pollutants. 
In this manner the source of the contamination is addressed, as opposed to 
identifying and then atteirpting to treat the surface water bodies which have been 
defiled. E. coli , P. aeruginosa and B. longum seem to all possess the criteria of 
having a short life. Therefore, for pfurposes of identifying the newness of fecal 
contaminants they could feasibly be employed. Hie experimental data collected 
indicates this conclusion, however, environmental concern regarding this topic does 
not end with sinply identifying how recent the pollution is, it also requires the 
differentiation of human and animal fecal contamination. The human input into the 
problem of surface water pollution may, with p^erseverance and regulation, be 
controlled; however, for the animal contribution this task would not be practical. 
Thus it becomes essential to discriniinate between human and animal fecal waste, 



- 158 - 

which as per the literature seems to necessitate the use of identifiable fecal 
streptococci . 

The second portion of this research study was involved with examining the 
bactericidal effect of chlorine upon E. coli . P. aeruginosa . S. faecium var. 
casseliflavus and B. breve . The wide acceptance of chlorine for purposes of 
disinfection stimulated interest in vy*iether or not chlorine, under appropriate 
conditions could be used as a sterilant. The fact that this research project began 
as a survival study must also not be overlooked. The two parameters v^ich were 
given consideration in this study included the pH of the environment in v^ich the 
chlorine functioned, as well as the particular concentration of the chlorine 
exposure dosage. The length of application of chlorine was not manipulated, being 
kept constant at one minute for cill trials. 

According to Caitper and McFeters (1979) e^qxDsure of waterbome organisms of 
fecal origin to hostile chemical environments results in a chain of events. The 
first of these is stress, which is then followed by injury and finally, death. If 
microorganisms have been exposed to chlorine, as in this experimentation, then the 
cultivation of survivors necessitates the use of a non-selective (not stressful) 
growth medium. For this reason, the antibiotic free nutrient, EHI, and MRS agars 
were used. In some cases it has been suggested that the injuries sustained due to 
chlorine may be reversed with the addition of appropriate metabolites to the growth 
medium (Heinmets et al., 1954). 

With reference to the Introduction, the biochemical effect of chlorine upon the 
bacterial cell may be found. One specific mode of operation, which was not 
mentioned previously, comes from the early work of Knox, et al . (1948) in which it 
was proposed that chlorine specifically oxidizes sulfhydryl groups of certain 
enzymes irrportant in carbohydrate metabolism. These hypothetical models of chlorine 



- 159 - 
action prc3vide a basis frcm which the physiologic understanding of this chemical's 
function may be extrapolated. This knowledge is essential if chlorine is to be 
considered for purposes of sterilization, since its limitations must be known. 

Experimentation with chlorine requires the use of acid-washed glassware, 
chlorine demand- free test waters and bacteria free of chlorine-demand products. 
Fitzgerald and Dervartanian (1969) presented experimental data, regarding P. 
aeruginosa , v*iich illustrated vAiy such materials are of paramount iirportance. An 
unwashed suspension of P. aeruginosa (concentration of 10E6) had a chlorine demand 
of 0.4 ppm v^le washed bacteria had a demand of one- tenth this amount. Some 
examples of chlorine-demanding products may again be found within the Introduction. 
The reason for their renoval prior to experimentation resides within their ability 
to stabilize chlorine and render it nearly functionless . 

One final point concerning the operation of chlorine deals with the kinetics 
and the more general functioning of this bactericidal agent. Three basic factors 
influence chlorine's effect on the bacterial cell. The first of these is the mass 
transfer of chlorine to the bacterial cell liquid interface. Secondly, the 
chemisorption of the chlorine at selective centres on the cell surface and finally, 
"the surface and intrasurface diffusion of the activated chemisorbed complex with 
attendant chemical attack on cellular elements" (Bemarde et al., 1967). 

This schemata illustrates the "gross" functioning of chlorine, as well, a feel 
for the numerous locations for rate limiting steps may be obtained. 

The experimental results obtained through this research project (see Results) 
seemed to correlate, with only two trial exc^jtions, to the theoretical concepts 
presented by Black et al. (1970), in vtiich it was reported that the bactericidal 
effectiveness of chlorine was greater at an acidic j^. The inplications here are 



- 160 - 
that the hypochlorite ion is not as effective a disinfectant as hydrochlorous acid, 
v*iich referring to Table 7, makes the adjustment of the pH a prerequisite for 
adequate killing. 



Table 6 



Effect of TpH on forms of Chlorine 





pH 




% 


CI 






% HOCl 




% OCl 




4 






0. 


.5 


99, 


.5 




5 













99.5 




0.5 


6 













96.5 




3.5 


7 













72.5 




27.5 


8 













21.5 




78.5 


9 













1 




99 



(Black et al., 1970) 

Further examination of the results shows that at higher concentrations the 
percentage of organisms killed was increased. The use of chlorine as a sterilant, 
however, is questionable. Neither the acidic conditions nor the increased 
concentration of 0.4 p^mi (theoretically ideal situation) provided 100% killing, 
demanded of an agent used for sterilization. B. breve was the only organism with a 
percentage killed close to that of sterility, and it was yet 2 percentage points 
short. Further increase in chlorine concentration, coupled with an eiqxssure time 
of greater than on minute may be needed to attain sterile conditions. 

The final aspect of the chlorine experimentation deals with the ability of some 
organisms to resist the lethal effects of this chemical. Four main factors are 



- 161 - 
associated with this resistance and they include: (i) cell surface structure 
mcdification vAiich may facilitate cell clurtping, (ii) bacterial adhesion to 
suspended particulate matter (clay particles), (iii) production of extracellular 
capsules or slime layers and (iv) formation of resistant spores (Ridgway and Olson, 
1982) . Frcm the test organisms used, P. aeruginosa was the only one with the 
ability to produce a slime layer which according to Brown (1975) may act by 
attaching chemicals and preventing their penetration into the cell. The 
experimental results in a pH of 9,0 do, for P. aeruginosa . indicate a lower 
percentage killed than the other organisms, hcwever, this was not the case at a pH 
of 6.0. P. aeruginosa ' s greater resistance may also be attributed to this 
organisms tendency to form cell aggregations. 



- 162 - 

REOCMMENDATICa^S 

6.1 Growth cycle determination 

To facilitate the study of the fecal streptococci a superior approach may 
be to ccmbine E. coli , P. aeruginosa . B. longum and the first case S. 
faecal is var. faecal is and in the second case S. faecium . 
The second alteration would be to study the growth of the organisms in an 
open-system, as opposed to the closed environment used in this study. 

6.2 Chlorine testing 

Ihe bactericidal effect of chlorine could be tested on more resistant 
organisms, such as the Mycoplasma or endospore forming organisms. 
Further experimentation with the manipultion of the time of e>:posure to 
chlorine, in conjunction with a wider range of chlorine concentration. 



- 163 - 

Appendix 



7.1 Buffers and Solutions: 



1) - Calcitnn hypochlorite (stock) solution: 

Ca(OC) 0.5g 
dH20 SOOmL 

Stir ingredients to dissolve. Refridgerate in the dark. 

2) - l.OM Hydrochloric acid: 

BDH Analar grade concentrated HCl 41.5iiiL 

dH20 SOOmL 

I' 

Add concentrated HCl to "200mL dH20 and then add the remaining 300inL dH20. 
Dispense into bottles and autoclave for 15 minutes at 121°C / 15 PSI. Ke^ 
ref ridgerated . 

3) - Phosphate solution: 

(A) Dissolve 34. Og KH2PO4 in 500mL dH20. 
Adjust pH to 7.2 

Dilute to 1 Litre with dH20 

(B) Dissolve 50g MgS04 • 7H2O in 1 Litre dH20. 

Autoclave both solutions separately for 15 minutes at 121°C / 15 PSI. Cool and 
store for up to 1 month. Add 1.25mL of (A) and 5mL of (B) to 1 Litre of dH20. 
Dispense as dilution blanks or for rinse water. Autoclave for 15 minutes at 
121°C / 15 PSI. 

4) - Sodium thiosulfate solution: 

Na2S203 0.35g 
dH20 SOOmL 



- 164 - 
Stir ingredients to dissolve. Add 5inL of Na2S203 to acid washed test tubes. 
Autoclave for 15 minutes at 121°C / 15 PSI. 

5) - l.OM Sodium hydroxide: 

UaCH 40. Og 

dH20 lOOmL 

Dissolve NaOH in dH20 slcwly. Dispense into bottles and autoclave for 15 
minutes at 121°C / 15 PSI. 

7.2 Growth Media; 

The following media was prepared according to manufacturer's specifications: 

DIPXD media: 

1) - BHI agar 

2) - Nutrient agar 1.5% (NA) 3) -Nutrient broth 

4) - Sugars for biochemical testing, prepared to a final concentration of 0.5%. 

- Arabinose - Cellcbiose - Xylose 

- Lactose - Raf f inose 

- Mannitol - Ribose 

- Melelitose - Sorbose 

- Melibiose - Trehalose 

5) - The remainder of the biochemical testing agents were also prepared to 
manufacturer's specifications: 

- Argenine - Litmus milk - Sodium chloride 

- Bile esculin - KLigler's - Todd Hewitt 

- Gelatin - Pyruvate 

GIBCD media 



- 165 - 

6) - MRS broth 

7) - MRS agar - with the addition of 0.03% cysteine 

hydrochloride 

8) - M-Enterococcus agar (m-Ent) : 

M-Enterococcus agar (DIFCD) 42g 

*Sterile dH2 IGOOmL 

*Autoclave distilled water first, prior to making media. Allow to cool. 
Weigh out agar in a sterile beaker using an alcohol flamed spatula. Heat to 
dissolve agar (93°C) . Cool rapidly to 60°C and dispense into square petri 
plates. Final pH 7.2 +/" 0-2. 
9) - m-E medium (Dufour's modified) : 

- Peptone 10. Og 

- Yeast extract 30. Og 

- Sodium chloride 15. Og 

- Sodium azide 0.15g 

- Actidione 0.05g 

- Agar 15. Og 

- dH20 lOOOmL 

Mix above ingredients and heat to 90°C. Autoclave for 15 minutes at 121°C / 15 PSI. 
Cool medium to 60°C and stir in *Antibiotic solution. Dispense into square sterile 
petri dishes. Store at 4°C after solidification. Final pH 7.1 +/~ O-l- 

♦Antibiotic solution: 
Add each separately, ascepticcilly 

A) Nalidixic acid - 0.240g in 3mL dH20 

and 0.2mL lOM NaOH 
Add last: 



- 166 - 

B) Triphenyltetrazolium chloride - 0.020g 

C) Indoxyl - B - D glucose - O.SOOg in 5inL 95% 

ethanol and SmL dH20 

10) - Ifedium for Pseudomonas aeruginosa (MPA) : 

- L - lysine roonchydroechoride 5,0g 

- Yeast extxact 2.0g 

- Xylose 2.5g 

- Sodium thiosulphate 5.0g 

- Magnesium sulphate, anhydrousl . 5g 

- Sucrose 1.25g 

- lactose 1.25g 

- Sodium chloride 5.0g 

- Ferric ammonium citrate 0.80g 

- Sodium desoxycholate O.lOg 

- Phenol red 0.08g 

- dH20 sterile 800mL 
Autoclave 800 mL distilled water before pr^aaring media. 

Mix above ingredients and adjust pH to 7.6. Add 15g agar. Heat to 93°C to 
dissolve agar and then cool to 60°C. Stir in *Antibiotic solution. Dispense 
into square sterile petri dishes. Store at 4°C after solidification. Final pH 
7.1 +/- 0.1. 

*Antibiotic solution: 

A) Sulfapyridine 0.1760g 

B) Kanamycin sulphate 0.0085g 

C) Naladixic acid 0.0370g 

D) Actidione O.lSOOg 



- 167 - 
Dissolve A to D in 200inL sterile dH20. Heat to 50°C to dissolve antibiotics. 

11) - Medium for the Isolation of Thermo Tolerant E. coli 
(M-Tec agar) : 



- Proteose p^jtone No. 3 


5.0g 


- Yeas=t extracrt 


3.0g 


- Lactose 


10. Og 


- NaCl 


7.5g 


- K2HPO4 ; - 


3.3g 


- KH2PO4 


l.Og 


- Sodium lauryl sulpiiate 


0.2g 


- Sodium deoxycholate 


O.lg 


- Brtsnocresol purple 


O.OSg 


- Bromo phenol red 


0.08g 


- Agar 


15. Og 



- dH20 

Mix above ingredients and heat to 90°C to dissolve agar. Autoclave for 15 
minutes at 121°C / 15 PSI. Cool to 50°C and dispense into sterile square petri 
dishes. Store at 4°C after solidification. Final jSl 7.1 +/- 0.1. 
12) - YN17 Blue (Used for the isolation of Bifidobacterium ) : 

- Yeast extract 20. Og 

- Polypeptone BBL 10. Og 

- Lactose 10. Og 

- Casamino acid 8.0g 

- Sodium chloride 3.2g 

- Bromocresol green0.30g 

- Cysteine hydrochloride 0.40g 



- 168 - 

- Agar 15. Og 

- dH20 lOOOltlL 

Mix above ingredients on medium heat until agar dissolves. Autoclave for 15 
minutes at 121°C / 15 PSI and then cool to 60°C. Stir in *Antibiotic solution. 
Dispense into sterile petri dishes. After solidification store at 4°C. Final 

pH 6.9 +/- O-l- 
*Antibiotic solution: 

A) Nalidixic acid 0.03g 

B) Kanaycin sulphate 0.05g 

C) Polymixin B 0.0062g 
13) - Growth media for Bifido YN-17 

- Follow same procedure as above, however, emit bromocresol green and 
antibiotic solution 



- 169 - 

REFERENCES 

American Public Health Asscxriation, American Water Works 

Association, and Water Pollution Control Federation. 1971. Standard Methods 
for the Examination of Water and Wastewater. American Public Health 
Association, Inc. , 13th ed. , New York. 

Bemarde, M.A. , Snow, W.B. , Olivieri, V.P. and Davidson, B. 1967. 

Kinetic and Mechanisms of Bacterial Disinfection by Chlorine Dioxide. Applied 
Micrctoiology. 15: 257-265. 

Black, A.P. , Kintnan, R>N. , Keim, M.A. , Smith, J.J. and Harlan, 

W.E. 1970. The Disinfection of Swimming Pool Water. Pari I. Conparison of 
Iodine and Chlorine as Swimming Pool Disinfectants. American Journal of Public 
Health. 60: 535-544. 

Black, A.P. , Keim, M.A. , Smith, J.J., Sykes, G.M. and Harlan, 

W.E. 1970. Ihe Disinfection of Swimming Pool Water. Part II. A Field Study 
of the Disinfection of Public Swimming Pools. American Journal of Public 
Health. 60: 740-750. 

Brodsky, M.H. and Schiemann, D.A. 1976. Evaluation of Pfizer 

Enterococcus and KH Media for Recovery of Fecal Streptococci from Water by 
Membrane Filtration. Applied and Environmental Microbiology. 31: 695-699. 

*Brown, M.R.W. 1975. The Role of the Cell Envelope in 

Resistance. Resistance of Pseudomonas aeruginosa , John Wiley and Sons: New 
York. (Ch. 3) . 

*Canper, A.K. and McFeters, G.A. 1979. Chlorine Injury and the 

Enumeration of Waterbome Coliform Bacteria. Applied and Environmental 
Microbiology. 37: 633-641. 

Doran, J.W. and Linn, D.M. 1979, Bacterial Quality of Runoff 

Water from Pastureland. implied and Environmental Microbiology. 37: 985-991. 

Fitzgerald, G.P. and DerVartanian, M.E. 1969. Pseudomonas 

aeruginosa for the Evaluation of Swimming Pool Chlorination an Algicides. 
Applied Microbiology. 17: 415-421. 

Geldreich, E.E. and Kenner, B.A. 1969. Concepts of Fecal 

Streptococci in Stream Pollution. Journal of Water Pollution Control 
Federation. 41: 336-352. 

Gibbons, N.E. and Buchanan, R.E. 1985. Bergey's Manual of 

Determinative Micaxbiology. The Williams and Wilkins Co.: Baltimore. 



- 170 - 

Gyles, C.L. 1984. Environmental Aspects of Escherichia coli in 

Human Health. National Research Council of Canada. NRCC. No. 22490. 

Heinmets, F., Taylor, W.W. and Lehman, J.J. 1954. The Use of 

Metabolites in Restoration of the Viability of Heat and Chemically Inactivated 
E. coli . Journal of Bacteriology. 67: 5-12. 

Isenberg, H.D. , Goldberg, D. and Saitpson, J. 1970. laboratory 

Studies with a Selective Enterococcus Medium. Applied Microbiology. 20: 433- 
436. 

Knox, W.E., Stunpf, P.K. , Green, D.E. and Auerbach, Y.H. 1948. 

The Inhibition of Sulfhydiyl Enzymes as the Basis of the Bacterial Action of 
Chlorine, Journal of Bacteriology. 55: 451-458. 

Lee, Wie-Shing. 1972. Improved Procedure for Identification of 

Group D Enterococci with Two New Media. Applied Microbiology. 24: 1-3. 

Levin, M.A. and Cabelli, V.J. 1972. Membrane Filter Technique for Enumeration of 
Pseudomonas aeruoginosa . ^^plied Microbiology. 24: 864-870. 

Levin, M.A. , Fischer, J.R. and Cabelli, V.J. 1975. Membrane 

Filtration for Enumeration of Enterococci in Marine Waters. ;^plied 
Microbiology. 24: 66-71. 

McFeters, G.A. . Bissonnette, G.K. , Jezeski, C.A. , Thomson, C.A. 

and Stuart, D.G. 1974. Cortparative Survival of Indicator Bacteria and Enteric 
Pathogens in Well Water. Applied Micrcbiology. 27: 823-829. 

Ridgway, H.F. and Olson, B.H. 1982. Qilorine Resistant Patterns 

of Bacteria from Two Drinking Water Distribution Systems. ;^plied and 
Environmental Microbiology. 4: 972-987. 

Ringen, L.M. and Drake, C.H. 1952. A Study of the Incidence of 

Pseudomonas aeruginosa from Various Natural Sources. Journal of Bacteriology. 
64: 841-845. 

Schuettpelz, D.H. 1969. Fecal and Total Coliform Tests in Water 
Quality Evaluation. MSc. Ihesis. University of Guelph. 

Seyfried, P.L. and Eraser, D.J. 1980. Persistence of Pseudomonas 

aeruginosa in Chlorinated Swimming Pools. Canadian Journal of Microbiology. 
26: 350-355. 

Seyfried, P.L. 1988. University of Toronto. PersoncLL 
Communication. 

Switzer, R.E. and Evans, J.B. 1974. Evaluation of Selective 

Media for Enumeration of Group D Str^stococci in Bovine Feces. Applied 
Microbiology. 28: 1086-1087. 

Taylor, R.H. , Bordner, R.H. and Scarpino, P.V. 1973. Delayed - 



- 171 - 

Incubation Membrane - Filter Test for Fecal Coliforms. ^plied Microbiology. 
25: 363-368. 

Wheater, D.W.F., Mara, D.D. , Lozan Jawad and Oragui. 1979. Journal of 
Biological Indicators of Waste Quality, John Wiley and Sons: New York (Ch. 
21) 



- 172 - 



APPENDIX C 

THE ISOIATICN AND lUtNl'lflCAnCN 

OF HEFIDCffiftCEERrA FPCM FBCAL 

AND SEHAGE SAMPLES 



In-ja Huh, Department of Microbiology 
University of Toronto 



- 173 - 

TABLE OF aCNEENTS 

Page No. 

ABSTRACT ^'^'^ 

INTRDDUCnON I'^S 

OBJECnVES ISO 

MATERIAI5 AND METHODS 181 

Collection and preparation of the test sattples 181 

Analysis of samples 181 

Identification of the isolates 182 

Carbohydrate fermentation profile 183 

Flash freezing 185 

RESULTS 185 

DISCUSSION ■ 154 

CONCLUSIONS 196 

RECCMMENDATIONS l^'^ 

APPENDIX 158 



REFERENCES 



202 



- 174 - 

T.T57 r OF TABCZS 

Table Page No. 

C-1 The carbohydrate fermentation profile used to 184 

speciate the bifidobacteria isolated from the 
fecal and sewage sanples 

C-2 Geometric mean concentration of FC, E^ coli 187 

and bifidobacteria in the storm and sanitary 
sewage samples 

C-3 Geometric mean concentrations of E^ coli ' 189 

and bifidobacteria in the feces of humans, 
cats and dogs 

C-4 The carbohydrate fermentation reactions 190 

of ATCC cultures 

C-5 The biochemical test scheme used for the 191 

identification of bifidobacteria in the 
profiles of ATCC cultures and their 
Gram Stain characteristics 

C-6 Species of bifidobacteria isolated from 192 

the two selective media that occurs in 
fecal and sewage sanples 



- 175 - 

LIST OF FIGURES 
Figure Page ^Jo. 

C-1 Geometric mean concentration of PC, E^ coli and 188 

bifidobacteria in the storm and sanitary sewage samples 

C-2 A conparison of the recovery of bifidobacteria species 193 

from feces and sewage on two selective media 

C-3 Percentage species of bifidobacteria in feces, sanitary 195 
and storm sewage 



- 176 - 

LIST OF ABBREVIATICNS 

° C = Degree Celsius 

FC = Fecal coliforms 

G = Grains 

hrs = Hours 

HBSA = Human bifid sorbitol agar 

i.e. = That is 

L = Litres 

LiCl = Lithium chloride 

MF = Membrane filtration 

mg = milligrams 

mL = Milliliters 

min = Minutes 

mm = millimeters 

m-Tec = Medium for thermotolerant Escherichia coli 

nm = Nanometres 

s = seconds 

YN-17 = Medium developed by Mara and Oragui for the enumeration of 
bifidobacteria 



- 177 - 

ABSTOACr 

The distribution of Bifidobacteria in the environment was studied 
utilizing two selective media: YN-17 and HBSA. The feces of humans, cats and 
dogs as well as sanitary and storm sewage samples were analyzed and 
Bifidobacteria were isolated in all cases except for the dogs. Species of 
sorbitol-fermenting Bifidobacteria, which have been previously reported as 
being exclusive to human hosts, were isolated from feces of human and cats as 
well as from sanitary and storm sewage sanples. However, 100% of the isolates 
from cats did not ferment sorbitol v*ien a carbohydrate profile was carried out. 
These sortitol-fermenting species were recovered using both YN-17 and HBSA. 
However, a higher percentage recovery was made on HBSA as this medium is 
specific for sorbitol-fermenting species. The results of this study indicate 
that sorbitol-fermenting Bifidobacteria may be useful as indicators of recent 
human fecal pollution in surface waters. 



- 178 - 

DracDDcrrcN 

cue to the disease hazards associated with fecally-contaminated water, it 
is imperative that the presence of fecal wastes be rapidly and accurately 
detected in surface water bodies, and the sources of these inputs traced in 
order to eliminate them. Thus a reliable bacterial indicator is desired which 
will detect recent fecal pollution and will in some way differentiate the 
source of the input (i.e. human and non-human) . . . .-. 

In 1981, the Toronto Area Watershed Management Study was initiated to 
better define the water quality conditions in Humber, Black Creek, Don Rivers 
and along Toronto Beaches. Particular emphasis was placed on urban areas of 
Metropolitan Toronto especially on point pollution inputs since high levels of 
fecal indicating bacteria were found in storm sewages. Subsequent studies 
began in the fall of 1982 to assess the contribution of pollution loading by 
the storm sewers and to trace the source of pollution in these lines. 
Unfortunately, pinpointing the original source of pollution is difficult 
because a specific bacterial indicator of human fecal wastes is not available 
at the present time. 

Traditionally E^ coli and FC have been used as the indicators of fecal 
pollution in storm waters and surface waters but due to its wide distribution 
in both human and animal feces they are not acceptable as an indicator of human 
input only. A number of workers have suggested using Bifidobacterium as a 
fecal indicator. (Buchanan and Gibbon, 1947; Scardovi et al, 1971; Levin, 1977 
and Resnick and Levin, 1981) . 

The genus Bifidobacterium includes gram-positive, non-sporulating, non- 
motile, anaerobic, pleomorphic rods. At the present time, there are 24 known 
species of bifidobacteria that have been identified. Bifidobacteria are 



- 179 - 
present in concentration of 10^ organisms per gram of feces in humans 
(Geldriech, 1978) , but have a very limited distribution among other animals 
(Mara and Oragui, 1983) . They also have been recovered from raw se'.^rage 
(Resnick and Levin, 1981) and in a Tropical Pain Forest Watershed B. 
adolescentis was isolated and enumerated. (Carillo et al, 1985) . 

The first synthetic selective medium that gave a reasonable recovery of 
bifidobacteria was invented by Gyllenberg and Niemen in 1960 and was called GN- 
6. This was modified in 1981 by Resnick and l£vin Vi^o added nalidixic acid to 
(3^-6 and named it YN-6. Ihen, in 1983, Mara and Oragui developed YN-17 medium 
which is more selective for bifidobacteria due to a further modification of YN- 
6 (i.e. addition of polymyxin B and kanaraycin sulphate) . These antibiotics 
decreased the streptococcal contamination in the selective medium but did not 
eliminate them. 

In that same study, Mara and Oragui proposed a methodology to detect 
bifidobacteria species as indicators of human fecal wastes in surface waters. 
They devised a medium called HBSA which is very similar to YN-17 except its 
replacement of fermentive carbohydrate lactose by sorbitol. Their method 
involved the isolation of bifidobacteria by membrane filtration of test sanples 
onto HBSA v^ch only allowed growth of sorbitol-fermenting species. This study 
reported that sorbitol-fermenting species of bifidobacteria were exclusive to 
human fecal wastes. These species were B^ adolescentis and B^ breve . 



- 180 - 

c«jBcrrvEs 

The purpose of this project was: 

To test the relative merits of HBSA in conjunction with another standard 

medium for the isolation and enimieration of bifidobacteria which is YlJ-17. 

To study the possible usage of MRS medium as a selective medium for 

bifidobacteria and test the effectivity of LiCl in repression of 

streptococcal contamination. 

Isolation and speciation of bifidobacteria found in feces of humans, cats 

and dogs as well as storm and sanitary sewage samples. 

To confirm vrtiether or not sorbitol-fermenting species are found in human 

waste but are absent in animal wastes. 

To compare the concentration of fecal coliforms and E^ coli with 

bifidobacteria count in feces and in sewage samples. 



- 181 - 

MATERIALS AND MEIHDDS 
Oollecticn and Pr^araticn of the Test Sanples 

In the fall of 1987 samples were collected from three points along a storm 
sewage line draining the Danforth-Pape Area and cilso from two points along a 
sanitary sewage line located in proximity to the storm water. The samples were 
collected in triplicates for statistical accuracy. This particular storm sewer 
line was chosen because it was suspected of having an illegal sanitary hook-up. 
Water samples were transported to the laboratory on ice in sterile sampling 
bottles with sodium thiosulphate to neutralize any chlorine that might be 
present. 

Fecal sanples were collected in presterilized wide-mouthed jars. A total 
of 20g of feces was weighed out, diluted 1 in 10 with sterile buffered 
phosphate water and mixed in a blender for 15s at low speed before being 
examined. All the samples were analyzed within 2-4 hours of its collection. 

Analysis of Sanples 

Both the sewage and fecal samples were tested by membrane filtration 
analysis. This method is one of the standard processes used in water quality 
monitoring (Cufour and Cabelli, 1975; Dufour et al, 1981). Serially-diluted 
samples were filtered through Gelman 0^-6 47mm nitrate filters having a 
porosity of 0.45um. A control sample of sterile buffered water was filtered 
for each of the samples by placing these filters on the least selective medium 
(i.e. m-Tec) . The filters of the sanples were planted on the selective media 
for the recovery of fecal coliforms and bifidobacteria. 

FC bacterial densities were determined by using m-Tec agar which was 
incubated at 44.5 +/~ 0-5° C for 23 +/" 1 h^^- The target colonies enumerated 



- 182 - 
were yellow, yellow-green and yellow-brown on this medium. To ensure the 
accuracy of the counts, filters with 10 - 100 target colonies were chosen to 
enumerate. These filters were subsequently tested for E^ coli confirmation 
using the urease test. The test involved placing the filter on a pad soaked in 
a urea phenol red solution and allowed to react for 15 minutes. Deamination by 
non-E. coli coliform bacteria possessing urease causes a colour change in the 
colonies from yellow to pink. A second count of urease negative (yellow) 
colonies was then taken. 

The isolation and enumeration of bifidobacteria was carried out using YN- 
17 and HBSA selective media. These media were incubated anaerobically in jars 
with hydrogen and carbon dioxide gas packs for 48 hrs at 37° C. The target 
colonies on the YN-17 medium were characteristically circular, convex, mucoid, 
2-3inm in diameter, glistening, greenish-blue and dark blue and on HBSA they 
were brownish-yellow to yellow. Streptococcal contamination on W-17 was 
exhibited by the presence of the greenish-blue colonies with pale green 
periphery an on HBSA by light yellow and clear colonies. These colonies were 
not enumerated and were considered as background. For the formulation of the 
media used see Appendix. 

Identif icatiai of Isolates 

From the total count of bifidobacteria that exhibited the typical target 
morphology on the selective media, 10% were isolated to be identified. These 
isolates were streaked out on a growth medium for purity. The growth medium 
used was YN-17 base without the antibiotics and the indicator. After 
incubation these isolates were Gram stained and those rods with typical 
pleomorphic morphology (i.e. bifurcated v an Y forms, club shaped ...) were 



- 183 - 
Streaked out for the second time on the growth medium for purity and incubated 
both anaerobically and aerobically. Isolates exhibiting either gram-negative 
rods or gram-positive cocci morphology were discarded. Only isolates which 
grew anaerobically were Gram stained once again and those with typical pleomorphism 
were considered as bifidobacteria. 

The confirmation of bifidobacteria was further verified by carrying out 
the following biochemical tests: catalase, gelatin and arginine, Kliger's ircn 
and bile esculin. Then the speciation of the bifidobacteria isolates -.vas 
accoiiplished by observing the fermentative reactions of the isolates in nine 
different sugars. The typical fermentation patterns of Bifidobacterium species 
is illustrated in Table 1. 

Carbohydrate Fermentation Profile 

The carbohydrate media was made by dissolving 8.0g of phenol red broth 
base in 450 mL distilled water. A filter sterilized sugar solution was added 
to this autoclaved broth to give final concentration of 1%. The sugars used 
were the following: L(+) Arabinose, D-Cellobiose, D-Mannitol, D(+) -Melezitose, 
D-Ribose, D-Sorbitol, soluble starch and D(+)Xylose. The test tubes of car- 
bohydrates were incubated for 24 hrs at 37 ' C before the inoculation of the 
isolates in order to reduce oxygen tension and to ensure sterility. The inoculated 
tubes were read after anaerobic incubation of 48 hrs and if the results were 
not definite, they were reincubated for an additional 24 hrs. 



- 134 - 
[•able C-1 



Tat]e !. The carbohydrate fermentation reaction profile used to 
sped ate the Bifidobacteria isolated from the fecal and sewage 
samples. { formulated from the Bergeys tlanual } 



BlfldODaCt. 


Arabnose 


Cellobiose 


Lactose 


Mannitol Mele:itose 


Ri bote Sorbitol 


Starc-h > 


'.ijIct- 


species 
















bifiijum 


- 


- 


+ 


- 


- 


- 


- 


lonaum 


+ 


- 


+ 


+ 


+ 


- 


V 


infanti? 


- 


- 


+ 


- 


+ 


- 


V 


bri=">'^ 


- 


V 


+ 


V V 


+ V 


- 


- 


8doli??ci?nti3 


+ 


+ 


+ 


V + 


+ y 


•♦• 


■^ 


ana upturn 


+ 


- 


+ 


- 


+ V 


+ 


+ 


catrnui-tiTijm 


+ 


+ 


+ 


i 

V 


+ * 


- 


•r 


P?ir'jdOC2tnijlatU 


m + 


V 


+ 


- 


+ V 


+ 


+ 


dentiijrn 


+ 


+ 


+ 


+ + 


+ 


+ 


f 


qlcibosum 


V 


- 


+ 


- 


+ 


+ 


V 


psijijdolciriqijm 


+ 


V 


V 


V 


+ 


+ 


■k 


cunii.-un 


+ 


- 


- 


- 


- 


+ 


+ 


choenrnjm 


- 


- 


+ 


- 


- 


•^ 


- 


ariirri.'jil;. 


+ 


V 


+ 


Y 


+ 


+ 


•T 


trirrTioC'Ciilum 


- 


V 


V 


V 


- 


+ 


- 


bourn 


- 


- 


V 


- 


- 


+ 


- 


maoriijrri 


+ 


- 


+ 


- 


+ 


- 


■^ 


pullorum 


+ 


- 


- 


- 


+ 


- 


+ 


3IJV?. 


+ 


- 


+ 


- 


- 


- 


■4. 


minirnij-n 


- 


- 


- 


- 


- 


^ 


- 


iub+iii? 


- 


- 


- 


+ 


+ J- 


4> 


- 


corurif-'''orme 


+ 


+ 


- 


- 


+ 


- 


- 


aStenoOt"; 


+ 


+ 


- 


- 


+ 


- 


f 


indicijrn 




+ 






+ 







"+"= The sugar was fermented 
■-"= The ?ugar va? not fermented 
■v'"= Some strai n? ferment this sugar 



- 185 - 
Flash Freezing 

The ATCC cultures were flash frozen for long-term storage. The cultures 
were prepared by growing on a growth medium for purity and quantity. Then 
under the Biohazard Hood, approximately 5 mL of 40% glycerol was poured onto 
each of the plates and with a flamed loop the colonies were dispersed. By 
using 1 mL pipettes the culture-glycerol solutions were soaked into small 
pieces of filter papers v*iich had been aseptically placed in labelled freezer 
bags. These were done in triplicates and were heat-sealed. The bags were 
placed in a small box and stored in a -70° C freezer. 

The frozen cultures were revived by planting the saturated filters on the 
growth medium and streaking for isolated colonies. 

Results 

The YN-17 medium allowed more accurate enumeration and isolation of 
bifidobacteria than HBSA as indicated by the higher percentage of false 
positive targets (i.e. 10% for HBSA and 5% for YN-17) . A large majority of 
these false positive targets were fecal streptococci. MRS agar with the 
antibiotic cocktail of YN-17 was also tested for its selectivity of 
bifidobacteria. Unfortunately, this medium supported growth of both 
streptococci and Lactobacillus species which resulted in higher concentrations 
of background bacteria. However, this medium shortened the incubation period 
by half and thus was used without the antibiotics as a growth medium. In 
addition, LiCl was incorporated at the concentrations of 0.3%-0.4% to YN-17 and 
HBSA; however it did not decrease the streptococcal contamination as was 
suggested by N.A. KLapes at the ASM meeting in March of 1987. 



- 186 - 

The geometric mean concentrations of FC, E^ coli and bifidobacteria in storm 
and sanitary sewage samples can be seen in Table 2. Also, a representative 
graph of the counts from storm sewage samples is illustrated in Figure 1. 
Both Table 2 and Figure 1 indicate that in storm sewers total bifidobacteria 
exist in hic^er concentrations than FC or E^ coli. The table and figure also 
show the trend towards decreasing concentrations of all the bacteria from 
points A to C due to dilution and die-off within the storm sewer line. From 
the graph one can clearly see that in storm se'^rage bifidobacteria exist in 
concentrations about 10 times greater than E^ coli . Note that there are 
higher concentrations of bacteria in sanitary se'.:rage than in stonn sewage and 
that variance in bacterial counts occurs over the different sample days. 

Results of the fecal analysis (Table 3) also illustrates a higher concentration 
of bifidobacteria than E^ coli (i.e. 10 - 100 times greater). Bifidobacteria 
were isolated from the feces of humans and cats but not from dogs. The sewage 
samples had lower concentrations of bifidobacteria and E^ coli than the fecal 
samples. This is due to dilution and to the fact that the organisms die off at 
a faster rate in the environment (Oragui, 1982) . 

The fermentative reaction of ATCC cultures of bifidobacteria (Table 4) 
closely corresponds with those of the Bergey's Manual profile used for speciation 
of the isolates (Table 1) . Table 5 illustrates the biochemical reactions used 
to confirm bifidobacteria (Bergey's Manual 9th ed.) . The ATCC cultures exhibited 
typical biochemical reactions for this genus (i.e. catalase (-) , arginine (-) , 
acid production in Kliger's iron medium and variable hydrolysis of bile esculin) . 

Recovery of bifidobacteria species from feces and sewage on two selective 
media is shown in Table 6 and Figure 2. A wider variety of bifidobacteria 



- 187 - 



Table C-2 



T^i^/e 2. osametnc mffs/? cance/itrdifa.'js of fees' Cij/!forj7?s. 
Esc^anc^/s ra// and Bffidcbscierld In the storm and ssnftsru 






( count;: oef 1 Ou mi of i^^rriDie ) 
( 1 9S6) its -SOU re* FC EC HESm 'iU-\ 



Oct Z: A 3933 3617 ■ - 1^-66 

Oct 26 A 10Z33 10200 133000 27000'; 

Nijv 13 A 39666 57500 21000 46000' 

3 26000 23700 1600 52000 

C 1 300 030 1 500 -000 

Oct 2o D 253000 ■ 220000 - - 96000 

Mcv 13 D 236 6000 



220000 


- 


1 700000 


f 7 ''! !'! r 


2600000 


4700:: 



E j7:.0iJiJU 

•J;2ri31 E - 2900000 42000 



'v'N-17 = Tijt.ii count of EifidOuoCtena 

H5'£a = Luunt of t.hi; iorDitoI ferment; no Bifidotj.icfen.^ 



- 133 



»— i 
—I 
O 
CJ 



LU 



cn 

O 
Ll_ 



O 



<: 

CJ 
LU 
Li. 

Ll_ 
O 

CD 






LU 
CJ 

O 
CJ 



LU 



CJ 
I— I 

tx 

LU 

O 
LU 
CD 



LU 
CD 

LU 

CJ^ 



a: 
a 



CL 
LU 

\— 

CJ 

<: 

CD 
O 

a 

Ll_ 
I— t 

02 

a 



^ >• 



i 
>• 



LU 






mo: 4. 

o as — 
a = 



cn I 



_1 LU 

O 1 

u 

LU 

LU 

II LU 






o ; 

u u. 

_I i 

< LL 
U 

LU , 

t 1 




I I I I I I I 





U3 


<- 




H- 






Z 






HH 


-0 


a 





2 




a. 


^ 


r— 


C3 


< 


N^ 


1: 




a 


I-* 


— ' 


a. 


—I 


z 




Q. 






Z 






•«t 


— i 




03 








! 5: 



o 
o 
o 



22 



q 



I 



(^W00;/W9) M0IiY«LNC3 "VIHrlGYS 



139 - 



Table C-3 

TaifiB J. Seomstric mas/? csncsnirdiians of ^•r.c.^.^^;r.?;.^ ,-.?.-/ art 
Sifidoiydcisria in tf^s faces af humans, cats and dags. 

S cure 8 EC H5Sa VN- \ 7 

( per I gr:m -jf r'^csi ) 

human 230.000,000 20,000,000 37.000 O'j-O 

cat 6,400,000 17,000,000 526.000,000 

dog 3,067,000 



- 'J30 - 



Table C-4 

Tj^/a 4. The chardci eristic ferinenisti'/e reset ions of 9 eugere 
by the A TCC cultures of Sifidottacteria. 



LiirjM.Ti 



iriT.KiTI! 



g!^r''';lor'jm + 



hrr^''* 



ir^nlrifprl'ltl ;: + + 



:ini3ij,vi 



- 191 - 



Table C-5 

TatJe 5. The bfochemica} issi scheme used for the 
fdentificatjon of Bifidohacieria in the profiles of A TCC culiur 
and their Sram Stain Characteristics. 



rrior'Dhc-loQ^j hudrolgsiJ hydro. h yd ''■::. ir:n 



I ^ ■»■ ~ f J L' ' ' I 'J '.i u I 





pliromorphic 




"■irripnor.-j- 




■ ike "Cell::; 


1^697 inf:5riti:5 


G +v* 




pltornorphic 


1 56 9S Dyrvulonjm 


G +ve 




branch! nq, 




lorig, ti-iui 




rods 


15700 tifrvf' 


G +ve 


15701 


thin.:?: .5 tort 




rods 


15702!1!je!-orurri 


G +ve 




loni] .i]^. short 




thin rod? 


15703.iijuir::.i::'!'nt:v 


G -^ve 



1 57 0~ piwrnorphio 

having many of 
other spirci's' 
morphology 

1 5707 long urn G +ve 

long cc'lls v/ith 
oiub ihapird ends 



'"^" - ir-'id produc'ion 

■'-■■= Hydrolysis v:3s odSc'rvi?d 

■■-"= Hudrolijjv? v.':s not obssrv^Jj 



- 192 - 
Table C-6 



Tall I a D. Species of difiuOudctsha isoJdted from the tYr-a 
ssJectfve media that occurs in fecal and sewape samples. 



3pc;C;r.;5 

j^:c^'r■^c^^^t1i; wuman 6/9 3/ 1 6 

«t 

jtorm sev^jQr - 4/4 

jynit^fu JcVtag* 1 ! /' i 1 O/I'i 

'one 'J"; riijn-ian l.-'9 7/16 

storm 

brr'-r fiurnsn 2/9 l/lo 

cat , 12/13 

storrn 

sanitary - 3/19 

gli^bcsum hurrian 

cat 

storm 

sanitary 1/13 ' 1/19 

D-re-iJcoc-atnji.-jtijm human 

car 1 / 1 3 

storm i/i 

sanitary 
suis human 

cat 

storm 

sanitary - 1/19 

ariQ ijii"'."' human 

oar 1/13 

storm 

sanitary 3/li 2/19 

thr-fTr;i;c;h;iijm nuTian 

cat 1 / 1 3 

sTorm 

sanitary 
'^I'lorium humian 

cat 

storm 

sanitary 
uniiientTTViie human 

cat 3/ i 3 

*tocm 

iamtjr-'j 2/13 2/19 



- 193 - 



O 

d 

cn 

I— I 

u 

LU 

Q- < 

cn H-( 
a 
<: LU 



cr I 

QJ LLi I 

h- > 

< I— 

CD CJ 

O IXl 

a —I 

j—l LU 

Ll_ 01 

m o 

U- I— 

o 

:^ o 

CL 

LU UJ 

> CD 

O < 

O S 

UJ LU 

cc o: 

LU a 

< ■ 

LU I 



a 
cn 
I— • 
cc 

-< 

Q. 

O 
CJ 



UJ 

< 

in 
cn- 



PS 






LU 
C3 

■< 



LU — rr 
cnr* 


>- 1 

crz 

<>- 



•< 
cn 



cn 

LU 
CJ 

Lu: 






cn 



CJ — 

Lur- 



zz 

■< >- 



I 




^^s 



cr 









<4.. 



I I I I I 



5fi:TC:>iV»:*f:*- 






% 






r 



^?????ff??t^^¥s^ 




cn 

LU 
hH 
U 
LU 

a. 
cn 






< 

1/0 



o 



! ^ 





< 


\ 


1 


'd. 


< 


y. 


(N 


<$> 


J, 








P 




H 








•H 


■>*. 








% 





53N=Hn330 39ViN53U3d 



c? 



- 194 - 
species were recovered in the sewage samples than in fecal sairples (Fig. 3) . 
Figure 3 illustrates that the predcminant species in human feces and se'^rage 
were B^ adolescentis and B^ breve which are the sorbitol-fermenting species of 
bifidobacteria (Mara and Oragui, 1983) . As indicated by Fig. 3 B^ breve was 
isolated from cat samples but 100% of these isolates did not ferment sorbitol 
when the carbohydrate profile was carried out. 

Discussicn 

The superiority of the two selective media is due to the antibiotic, 
kanamycin sulphate, which reduced levels of the contaminating streptococci 
greatly v^en used in the specified concentration. However, it didn't 
completely eliminate the background of streptococci. Yield of bifidobacteria 
was higher on YN-17 than PiBSA because HBSA was a more selective medium in that 
it only allowed growth of sorbitol-fermenting species. In addition, target 
colonies were more easily distinguished on YN-17, while there was a tendency 
towards more false (+) targets arising from streptococci on HBSA. 

The data presented herein disagreed with the study done by Mara and Oragui 
in 1983. In this project it was discovered that their description of 
Bi f idobacterium on YN-17 (i.e. blue centre with pale green periphery) was in 
fact a mixture of bifidobacteria and streptococci. Thus, during the beginning 
of the year the successful recovery rate of bifidobacteria was considerably 
lower; however, accuracy of 95% and higher was achieved during the latter part 
of the year. 

Bifidobacteria can be used as fecal indicating bacteria because they exist 
in high number in both feces and fecally contaminated environments. They also 



135 - 



f I I 



a 
•z. 
<: 

>- 

<: 



en 



en 

LU 
CJ 
LU 
Ll. 



LU 5 



en 



CD 
U- 

o 
en 

LU 

I— I 

CJ 
LU 

a. 
en 

LU 

eD 



ILU 

cr 

LU 
C 



o 



C31 



UJ 

■< 
X 
Ui 

en 



c 






"^ 



CO 



Ui 

cs 

< 

UJ 

en 

>• 
cr 
■< 



•< 

03 



\ 
\ 

^ 






>i. 



^ 



H' 



SJ 



d 



Essss: 



[Id 



en 

UJ 

u 

UJ 

u. 

z 



I 






% 






IP. 









>. 



\\\\\\\\\\\\\\\\^\\^y\\\\^\' ^■ 



o 

IS 



en 



u 

UJ 

a. 
en 




! C3 

z 

< 



I <: 






■ s 

i 3 



1 -^ 



I '-J 



I < 



a. 

m 
I 



b 



S3N2fcinC30 39ViN=2UHd 



- 196 - 
do not multiply outside the body and a rapid methodology for recovery from the 
aquatic environment is available as illustrated in this study. 

Site A of the storm sewer line is the likely location of the illegal 
connection since the concentration of fecal indicating bacteria was highest at 
this in-line point. The illegal hook-up is also confirmed by the presence of 
sorbitol-fermenting bifidobacteria in the line. Sanitary sewage samples 
contained a wide variety of species of Bi f idobacterium which is possibly due to 
some inputs of animal wastes as well as humans, but the highest recoveries made 
were again of sorbitol-fermenting bifidobacteria and this correlates with the 
face that human feces mate up the greatest percentage of wastes in sanitary 
sewage. Although bifidobacteria can be recovered from cats, it is unlikely 
that cat feces represent a major source of fecal contamination in the storm 
sewer line. Therefore sorbitol-fermenting species can be used to monitor 
recent human fecal contamination in sewage lines. 

Oonclusicns 

* YN-17 yielded higher concentrations and represented a broader spectrum of 
different species of bifidobacteria than HBSA. 

* B^ adolescentis was the most prevalent species of bifidobacteria in human 
feces and sewage samples, but were absent in cats. 

* Sorbitol-fermenting species (i.e. B^ breve) were isolated from both human 
and cat feces but 100% of the isolates from the cats didn't ferment 
sorbitol when the carbohydrate fermentation profile was carried out. 

* Bifidobacteria counts were 100 times greater than E^ coli in feces and 10 
times greater in sewage sanples. 



- 197 - 
Recannendaticns 

* Further study to confirm the specificity of the selective media for 
bifidobacteria. 

* Carry out a large scale fecal sairple study to see if the sorbitol- 
fermenting species, B^ adolescentis , is predominant in human feces and is 
limited or absent in animal feces. 

Acknowledgements 

The author would like to sincerely thank Elizabeth Harris and 
Dr. P.L. Seyfried for their support throughout the year. 



- 198 - 

1. Dissolve 34. Og of KH2PO4 in 500 mL distilled water and adjust the pH to 
7.2. Then dilute this to IL by adding distilled water. 

2. Dissolve 50 g of M3SO4 7H2O in IL of distilled water. 

These two solutions were autoclaved separately for 15 min at 121° C. After 
these were cooled to room temperature, add 1.25 mL of 1) and 5 mL of 2) to XL 
of distilled water. Then it can be dispensed either as dilution blanks or as 
rinse water for M.F. and autoclave again at 121° C for 20 minutes. 



- 199 - 
m-Tec TWgar 

This medium is used to isolate and enumerate the Fecal Col i forms as well 
as the Thermo-tolerant Ej. coli . 

Proteose peptone #3 10.00 g ■ 

Yeast extract • 3.00 g 

Lactose 10.00 g 

NaCl 7.50 g 

K2HPO4 3.30 g 

KH2PO4 1.00 g 

Sodium lauryl sulphate 0.20 g 

Sodium deoxycholate 0.10 g 

Bromocresol purple 0.08 g 

Bromophenol red 0.08 g 

Agar 15.00 g 

Distilled water 1.00 L 
The above ingredients were mixed and heated to dissolve. Autoclaved for 

15 min at 121° C and dispensed into Petri dishes after it is cooled. Before 

pouring the pH is adjusted to 7,1 +/~ O-l- ' 



- 200 - 
Bie two selective media for Bifidobacteria 

Yeast extract 

Poly-peptone (BBL) 

Lactose 

Casamino acid 

NaCl 

Bromocresol green 

Bromocresol purple 

Cystein hydrochloride 

Agar 

Distilled water 

The above ingredients were mixed in a 6L beaker and were stirred on medium 
heat until agar was dissolved. Then it was autoclaved for 15 minutes at 
121" C. After the isolation was cooled to 60" C the following were added: 
Nalidixic acid 30.0 mg 20.0 mg 

Kanamycin sulphate 50.0 mg 50.0 mg 

Polymixin B 6.20 mg 1.20 mg 

Finally, the pH was adjusted to 6.9 +/- 0-1 a^ then the media were poured 
into square Petri dishes (100 on^) . 



YN-17 




HRSA 


20.00 


g 


20.00 g 


10.00 


g 


10.00 g 


10.00 


g 


10.00 g 


8.00 


g 


8.00 g 


3.20 


g 


3.20 g 


0.30 


g 


- 


- 




0.10 g 


0.40 


g 


0.40 g 


15.00 


g 


15.00 g 


1.00 


L 


1.00 L 



- 201 - 
Distinct Gram MorphDlogies that are Species Characteristics of Bifidobacteria 



Bifidobacterium species 



Gram morphology 



bifidum 



loncrum 



breve 



ancrulatum 



catenulatum 



qlobosum 



animal is 



hi<^ily variable but has the typical 
"airphora-like" shaped cells 

very elongated and relatively thin 
cell with slightly irregular contours 
and rare branching 

thinnest and shortest rods 

v( angular) or palisade arrangement 
similar to those of corynebacteria 

chains of 3, 4 or more globular 

elements 

distinct branching 

short, coccoid or almost spherical to 
curved or tapered 

arranged in singly or doubly or rarely 
in short chains 

characteristic central portion slightly 
enlarged 



- 202 - 

ReferEncjes 



1. Ali, M. , Murray, P.R. , Sondag, J.E. 1979. Relative Recovery of anaerobes 
on different isolation media. J. of Clinical Microbiol. 10(5) . 

2. Cabelli, V.J. , Dufoixr, A. P., McCabe, L. J. , Levin, M.A. 1983. A marine 
recreational water quality criterion consistent with indicator concepts 
and risk analysis. J. of Water Poll. Cont. Fed. 55: 1306-1314. 

3. Carillo, M. , Estrada, E. , Hazen, T.C. 1984. Evaluation of 
Bifidobacteria as a possible indicator of human fecal contamination in 
tropical fresh-water. Abstract from the Annual Meeting of the American 
Society of Microbiology, March 4-9, 1984. 

4. Carillo, M. , Estrada, E. , Hazen, T.C. 1985. Survival and enumeration of 
the fecal indicators Bi f idobacterium adolescentis and Escherichia coli in 
a tropical rain forest watershed. ^^1. Environ. Microbiol. 5 0(2): 468- 
476. 

5. Cufour, A. P., Strickland, E.R. , Cabelli, V.J. 1981. Membrane filter 
method for enumerating E^ coli. Appl. Environ. Microbiol. 41(5): 1152- 
1158. 

6. Cufour, A. P., Cabelli, V.J. 1975. A membrane filter procedure for 
enumerating and component genera of the coliform group in seawater. Appl. 
Microbiol. 29: 319-833. 

7. Essers, L. 1982. Simple identification of anaerobic bacteria to genus 
level using typical antibiotic susceptibility patterns. J. of Appl. Bact. 
52: 319-323. 

8. Geldreich, E.E. 1970. Applying bacteriological parameters to 
recreational water quality. J. Amer. Wat. Works Assoc. 62(2): 113-120. 

9. Geldreich, E.E. 1978. Bacterial populations and indicator concepts in 
feces, sewage, stormwater and solid wastes. 

10. Gyllenberg, H. , Niemella, S., Sormunen, T. 1960. Survival of bifid 
bacteria in water as compared with that of coliform bacteria and 
enterococci. Appl. Microbiol. 8:20. 

11. Mara, D.D. , Oragui, J.I. 1983. Sorbitol-fermenting bifidobacteria as 
specific indicators of human fecal pollution. J. of Appl. Bact. 55: 349- 
357. 

12. Mutai, M. , Tanaka, R. 1980. Improved medium for selective isolation and 
enumeration of Bifidobacteria. Appl. Environ. Microbiol. 40(2): 866-869. 

13. Oragui, J.I. 1982. Bacteriological methods for the distinction between 
human and animal fecal pollution. Eh.D. Thesis, University of Leeds, 
England. 

14. Resnick, I.G., Levin, M.A. 1981. Quantitative procedure for enumeration 
of Bifidobacteria. Appl. Environ. Microbiol. 42(3): 433-438. 



- 203 - 

APEBOUX D 

A STUDY OF "fflE SURVIVAL OF BIFIDOBACIERIA AND 
"fflEIR RDLE IN WKEEK QUALITY CUNIMDL 



Sheila Shibata, Department of Microbiology 
University of Toronto 



- 204 - 

TABLE OF OCNTENIS 

Page No. 

LIST OF TABLES 205 

LIST OF FIGURES 206 

LIST OF ABBREVIATIONS 207 

INTRDDUCnON 208 

OBJECTIVES 211 

METHODS AND MATERIALS 212 

PART I: ISOLATION AND ENUMERATION 212 

Sample Collection 212 

Sample Preparation 212 

Selective Media 212 

Enumeration 214 

Identification 214 

PART II: SURVIVAL NETOORK 215 

Assembly and use of Chambers 215 

Preparation of Pure Cultures for 215 

use in Diffusion Chambers 

Preparation of Feces for use in 217 

Diffusion Chambers 

Sanpling of Chambers 217 

RESULTS 219 

PART I: ISOLATION AND ENUMERATION 219 

PART II: SURVIVAL STUDY 219 

DISCUSSION 230 

PART I: ISOLATION AND ENUMERATION 230 

PART II: SURVIVAL STUDY 231 

CONCLUSIONS 234 

RECOMMENDATIONS 234 

APPENDIX 1: BUFFERS AND SOLUTIONS 235 

APPENDIX 2: GROWTH MEDIA 236 

REFERENCES 238 



- 205 - 

UST OF T AFtrry; 

Table Page Mb. 

D-1 Source of envirormiental water used in vitro for the 217 

study of bifidobacteria! survival. 

D-2 Conparison of levels of bifidobacteria isolated from 220 

human feces on YN-17 and MFN. 

D-3 Levels of bifidobacteria and E^ coli in human feces 221 

as isolated on YN-17 and m-TEC. 

D-4 Levels of sorbitol-fermenting bifidobacteria and E^ coli 222 
in Lake Ontario water enumerated over a maximum three 
day period. 

D-5 Mean survival counts of bifidobacteria and E^ coli in 223 

Lake Ontario water enumerated over a maximum three 
day period. 



- 206 - 

IiLST OF FIGURES 
Figure Page No. 

D-1 Dialysis membrane diffusion chamber. 216 

D-2 In vitro survival of bifidobacteria isolated from 224 

sewage and Ei coli in Lake Ontario water. 

D-3 In vitro survial of bifidobacteria isolated from 226 

human feces and E^ coli in Lake Ontari water. 

D-4 In vitro survival of Bifidobacterium bifidum 227 

(ATCC #696) and E^ coli in Lake Ontario water. 

D-5 In vitro survival of Bifidobacterium breve 2 2 3 

(ATCC #701) and E^ coli in lake Ontario water. 

D-6 In vitro survival of fecal bifidobacteria and 229 

E. coli in Lake Ontario water. 



ATCC 

dH20 

g 

h 

HBSA 

min 

niL 

m-TEC 
MEN 

MRS 

YN-17 

YII-17(-) 



- 207 - 

UST OF ABBREVIATICNS 

American Type Culture Collection 

distilled water 

grains 

hours 

Human Bifid Sorbitol Agar 

minutes 

milliliters 

Medium for selection of thermotolerant E^ coli 

Modification of Petuely's PMS for the isolation of 
bifidobacteria 

Ifedium for the selection of lactobacillia used also for 
isolation of bifidobacteria 

Medium for the isolation of bifidobacteria 

Non-selective medium used to support the growth of 
bifidobacteria (Modification of YN-17) 



- 208 - 

The relatively recent closure of a number of Toronto beaches due to fecal 
pollution signifies a need to improve water quality in this area. Outbreaks of 
various infections traced to fecal contamination of surface waters are well- 
documented (Dart and Stretton, 1980) . 

Development of a management strategy plan to reduce fecal contamination 
requires suitable detection methods that can locate fecal inputs and 
differentiate between human vs. non-human sources of pollution. 

Ideally the enumeration of pathogen concentrations in surface waters would 
serve to indicate levels of contamination and potential health hazards. 
However, pathogens pose problems due to their sporadic occurrence in the 
environment and the difficulty in their collection and isolation. Hence, a 
more suitable surrogate indicator organism found in feces must be employed. 

Geldreich, in 1968, and Bonde, in 1983, have proposed a number of parameters 
for an ideal water quality indicator. Briefly stated: i) the indicator should 
exist at density levels far exceeding pathogen concentrations in feces of 
infected individuals; ii) the indicator should only be present in warm- 
blooded animals; iii) there should exist a relatively rapid and inexpensive 
means of detecting and enumerating the indicator; iv) indicator's survival 
characteristics should parallel those of the pathogen (s) . 

The most widely accepted indicator of water quality irrpairment due to human 
and animal excrement is the fecal coliform count. The Ontario Ministry of the 
Environment utilizes a standard of 100 fecal coliforms/100 mL of water as their 
upper limit of safety (MacDonald, 1986) . However, this indicator has a 
drawback in that it enumerates Klebsiella which is not restricted to feces 
(MacDonald, 1986) . 



- 209 - 

Escherichia coli is also being utilized as an indicator of fecal 
contamination in surface waters. Unlike other fecal coli forms it has not been 
isolated from non- fecal sources. Due to its fairly rapid die-off in situ . E. 
coli has been enployed as an indicator of recent feccil contamination (Seyfried 
et al . , unpublished report) . 

Unfortunately, the fecal coliforms, including E^ coli , when used as water 
quality indicators, are incapable of distinguishing between human and animal 
sources. These organisms are ubiquitously found in higher animals. 

Mossel (1958) was the first to propose bifidobacteria as potential 
indicators of fecal contamination. Bifidobacterium is a gram-positive, 
anaerobic, non-sporulating, non-motile rod often displaying pleomorphic forms 
(i.e. Y and V-shaped bifurcated rods) . 

Several media have been proposed for the isolation of bifidobacteria. In 
1983, Mara and Oragui developed YN-17 medium stating relatively good 
selectivity for bifidobacteria. Tanaka and Mutai (1980) stated similar results 
with the use of MFN medium, a modification of Petuely's Synthetic Medium (PSM) 
(Petuely, 1956). MRS, a medium normally utilized for the isolation of 
Lactobacillus spp. has been enployed for the isolation of bifidobacteria by the 
Department of Nutrition at the University of Toronto (personal communication) . 
Most contamination reported on these three media was the result of fecal 
streptococci growth. Lithium chloride was found to control unwanted growth of 
Streptococcus spp. . It was found to exert an inhibitory effect at 
concentrations of 0.3% and 0.4% in MSI5M Staphylococcus aureus medium (N.A. 
KLapes, Annual Meeting, American Society of Microbiologists 1987) . Its 
effectiveness in selective media for bifidobacteria has yet to be investigated. 



- 210 - 

It has been suggested that bifidobacteria could be used to distinguish 
between human and animal fecal pollution (Buchanan and Gibbons, 1974; Levin, 
1977; Cabelli, 1979; Resnick and Levin, 1981b). However, it was not until 1933 
that a method was devised by Mara and Oragui that would allow bifidobacteria to 
serve this purpose. Using a sorbitol -based medium (HBSA) , they isolated and 
analyzed bifidobacteria from human and animal feces. Their findings showed 
that certain sorbitol-fermenting species were restricted to human feces. 

The usefulness of bifidobacteria as a specific indicator of human fecal 
pollution could be limited due to its short survival in the extra-intestinal 
environment (Resnick and Levin, 1981; Oragui, 1982). Survival studies have 
shown bifidobacteria to die off more rapidly than E^ coli upon exposure to 
environmental waters (Carillo, Estrada and Hazen, 1985) . A too rapid decline 
of bifidobacteria in the aquatic environment and subsequent inability to detect 
their presence could render the organisms poor indicators of fecal contamination. 

In 1972, McFeters and Stuart devised dialysis membrane filter chambers for 
in situ and in vitro survival studies. The chamber allows for maximum exchange 
of molecules (e.g. gases, organic nutrients, toxic chemicals) between the 
environment and a pure culture of bacteria without contaminating the culture by 
introducing new water-borne bacteria into it. 

Before bifidobacteria can be used as an indicator of recent human fecal 
contamination in the Toronto area, their survival in this environment and their 
ability to satisfy the parameters of a good indicator must be investigated. 



- 211 - 
OBJECTIVES OF RESEARCH 

1. To assess the suitability of three media: YN-17, MFW, and MRS; in their 
selectivity for bifidobac±eria. 

2. To enumerate bifidobacteria (all species), bifidobacteria (sorfcitol- 
fermenting) and Ej. coli from human feces. 

3. To test the inhibitory effects of lithium chloride on bifidobacteria and 
fecal streptococci utilizing YN-17. 

4. To conpare the survival characteristics, in vitro , of mixed cultures and 
laboratory isolates of bifidobacteria with those of E^ coli . 

5. To investigate the suitability of bifidobacteria as indicators of recent 
human fecal pollution. 



- 212 - 

ME3IECS AND MftlERIAIS 
I^rt 1: Isolatlcai and Enumeratlcai 
SAMPLE COLLECYlOtf 

Feces were collected from healthy individuals, in sterile, wide-mouthed 
glass jars. All sainples were freshly voided and usually examined in the 
laboratory within four hours of defecation. 
SAMPLE FREPARAnai 

A total of 20g of each fecal saiiple was weighted out and transferred to 
sterile glass blender jars which had been calibrated previously to a 200niL 
volume. Sterile phosphate solution (Appendix 1) was added to the 20CrmL volume 
mark producing a 10"^ dilution of the original sample. The diluted samples 
were homogenized for 15 seconds at low speed vising a Waring blender. 
Systematic 100-fold dilutions were made of the blended samples in sterile 99mL 
dilution blanks (Appendix 1) prior to membrane filtering. 
Membrane Filtraticn 

Dilutions of the fecal samples were membrane filtered according to the 
methods outlined in Standard Methods for the Examination of Water and 
Wastewater (1980). The filtrations were performed utilizing Gelman 0^6 47ram 
cellulose hitrate filters with a porosity of 0.45um. Filters were planted on 
the appropriate selective media. For each sample a control was run on the 
least selective medium viiich was usually m-TEC. 
SEIECnVE MEDIA 

m-TEC medium (Dufour, 1975) and the Urease Test (Dufour, 1981) were employed 
for the selection and enumeration of E^ coli from the samples tested. The 
medium was incubated aerobically for 24h at 44.5° C in the presence of two 
plastic containers, holding 50mL of ice each, to ensure a resuscitation step 



- 213 - 
necessary for target colony growth. The m-TEC medium (Appendix 2) is 
selective for all fecal coliforms present in the samples. Target colonies of 
all yellow, yellow-green and yellow-brown colonies were enumerated as the fecal 
coliform count. To differentiate E^ coli from other thenrotolerant fecal 
coliform bacteria, the Urease Test was employed. The sanple filter was removed 
from m-TEC and placed onto a sterile filter pad saturated with a urease 
solution (Appendix 1) . All urease-negative (yellow) colonies were recorded as 
the Ei coli count. . 

The MEN medium formulated by Tanaka and Mutai (1980) was utilized to select 
for bifidobacteria. MEW medium (Appendix 2) was incubated anaerobically at 37° 
C for 48h with GasPaks (BBL) in an atinosphere of 95% hydrogen and 5% carbon 
dioxide. Target colonies were round, l-2mm in diameter, convex, flat with an 
entire edge and creamy vi*iitish-yellow in appearance. 

MRS medium (Appendix 2) was proposed as being selective and specific for 
bifidobacteria (Department of Nutrition, University of Toronto, 1987) . The 
medium was incubated anaerobically at 37° C for 24h with GasPaks (BBL) in an 
atmosphere of 95% hydrogen and 5% carbon dioxide. Presumptively positive 
bifidobacteria colonies were those that were circular, 2-3ram in diameter, dark 
green or blue, convex, and mucoid. Fecal streptococci contamination was 
observed as the presence of colonies l-2mm in diameter round and green with a 
pale green periphery. 

The YN-17 medium (Appendix 2) formulated by Mara and Oragui (1983) was also 
utilized for its selectivity and specificity for bifidobacteria. The medium 
was incubated anaerobically for 48h employing the same anaerobic techniques 
used for MRJ and MRS media. Target colonies and background contamination were 
identified by the same criteria used for the MRS medium. 



- 214 - 

Human Bifid Sorbitol Agar (HBSA) (Appendix 2) was formulated by Mara and 
Oragui (1983) . The medium was used to select for sorbitol-fermenting 
bifidobacteria. Incubation procedures were consistent with those used for YN- 
17 medium. Target colonies were taken as those colonies that were deep yellow 
and brownish-yellow, 2-3ram in diameter, circular and convex. Contamination of 
fecal streptococci were those colonies measuring l-2mm in diameter, flat, pale 
yellow or colorless. 

Concentrations of 3% and 4% LiCl were incorporated into a separate batch of 
YN-17 media to test for suppression of fecal streptococcal growth. Incubation 
procedures and target and non-target identification criteria were consistent 
with those utilized for the YN-17 medium. 
ENUMERATICN OF BTFTDOBACTERIA AND E. CPU 

Human fecal sanples were obtained and processed as mentioned above. 
Enumeration of bifidobacteria and E^ coli was performed using the membrane 
filtration method and the two media, m-TEC and YN-17. 
JEEtTCIFlCfinCtl OF BIFIDOBACrERIA 

Colonies from YN-17, ME^, and MRS were picked and streaked on YN-17 (-) 
(Appendix 2) to purify. Isolates were classified as gram-positive or gram- 
negative organisms. Gram-positive rods exhibiting the typical pleomorphic 
morphology were restreaked and incubated in aerobic and anaerobic environments. 
Those isolates growing anaerobically were presumed to be bifidobacteria and 
were subjected to further biochemical tests. The confirmation was performed by 
Inja Huh (1986-87) using the following biochemical tests: catalase, gelatin, 
arginine, bile esculin, and KLiger's iron. Further identification of the 
isolates to the species levels was accomplished using carbohydrate fermentation 
tests (Huh, 1986-87) . 



- 215 - 
Part U: Survival Study 

ASSIMBKir AND USE OF DIFHJSiai OffiMEERS 

SOmL dialysis mentorane diffusion chambers used in the survival study were 
designed by McFeters and Stuart (1972) . The chamber consists of a three-part 
plexiglass II cell with membrane filter sidewalls (Fig. 1) . Filter sidewalls 
were made of a polycarbonate film with a 99mm diameter and a porosity of 0.2um 
(Nucleopore) . Filters were placed between the plexiglass outer walls and the 
centre piece. Wing nutbolts were loosely set in place to hold the assembled 
chambers together. The chambers were autoclaved for 25min. at 121° C (ISlbs 
pressure) . Before use, the wing nutbolts were tightened. 
E5?EPARATICN OF KIKE BACTERIAL CUIIIURES PCR USE IN DIFFUSiaJ CHAMBERS 

Pure cultures of bifidobacteria and E^ coli were prepared by inoculating 5mL 
of MRS broth (Appendix 2) with a single colony from a pure culture of bacterium 
grown overnight on YN-17(-) (Appendix 2). The broth cultures were incubated 
for 48h at 37° C. Broth cultures were subjected to centrifugation at 300 rpm 
for 10 min. The supernatant was discarded and the pellet resuspended in 5mL of 
chilled gelatin phosphate buffer (Appendix 1) and recentrifuged. This step was 
repeated twice. After the supernatant was discarded, the cells were 
resuspended once again in 5mL of sterile gelatin phosphate buffer and measured 
spectrophotometrically at 660nm. For bifidobacteria an optical density reading 
of greater than 1.0 signified a culture content of approximately 10^ bacterial 
cells. For E^. coli . an optical density reading of greater than 1.1 signified a 
culture content of approximately 10^ bacterial cells. Further preparation of 
the cultures was performed as follows: 



- 216 



Figure D-1 



DIALYSIS DIrFUSIOri CHAiOZR 



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(McFeters i Stuart, 1. 



- 217 - 

i) Tenfold dilutions of the culture were made up to a 1 in 10^ dilution of the 
original culture in 99inL dilution blanks (Appendix 1) . The dilutions were 
analyzed by meinbrane filtration and the filters placed onto appropriate 
selective media (i.e. m-TEC for E^ coli and YN-17 for bifidobacteria) . The 
media were incubated according to the mEdium and genus requirements, as 
earlier stated, and subsequent colony counts were obtained to calculate the 
cell density of the original pure culture. 

ii) A 1 in lO'hiiL dilution was used to fill the chaii±>ers so that the 
approximate cell density would fall between 10'^ and 10^ organisms per 
mL. This dilution was achieved by transferring 3mL of the 1 in lO^mL 
dilution to a flask containing 297mL of sterilized environmental water. 
Table 1 presents a list of the locations from which the environmental 
water was obtained for each culture utilized in the survival study. 
The sterile, assembled chambers were inoculated with the pure bacterial 
culture using a sterile 50mL luer-lock syringe. The chambers could 
accommodate slightly more than the specified volume and were therefore 
filled to capacity. The chambers were then placed in a glass tank 
containing environmental water at room temperature. 

FREPARATiai OF FECES FOR USE IN DIFFUSION CHAMBERS 

A human fecal sample was obtained and prepared as outlined in Part I, 

Materials and Methods, of this report. The sample was diluted to a 1 in 10%L 

dilution and 3mL of this dilution were placed in 297mL of sterilized 

environmental water. Chambers were filled as stated above. 

Table 1 

' SOURCE OF ENVIRONMENIAL WATER USED IN VITRO 
FOR THE STUDY OF BIFIDOBACTERIAL SURVIVAL 

ENVIRONMENTAL WATER 
LOCATION 

lake Ontario: Harbourfront 
Queen City Yacht Club dock 

Lake Ontario: at foot of Brimley Road 

Lake Ontario: Kew Beach near Woodbine 
Race Track 



IDENl'lFICATTON 


TYPE OF ISOLAT] 


CODE 




308 


fecal isolate 


701 


B. breve 


696 


B. bifidum 


P-Z 


feces 


EY-6 


sewage isolate 



- 218 - 
SAMPUNG OF CHAMBERS 

The chambers were sanpled at 2, 5, 12, 24, 48, and 72 hoiars using lOmL 
disposable luer-lock syringes. The syringe was locked onto the port possessing 
the attached capillary tube and pumped five times to resuspend settled cells 
prior to removing the sample. Volumes from 1 to 12mL were removed, 
appropriately diluted, membrane filtered, and planted on both YN-17 and m-TEC. 
Bacterial densities were enumerated after incubation. 

NOTE: All samples for the survival study were performed in triplicate. 



- 219 - 

RESunrs 

Part 1: Isolatlcn and Enumeratlcn 

Of the three media tested for their selectivity and specificity for 
bifidobacteria from human feces, MRS displayed a high density of contamination. 
Colonies obtained included streptococci, and lactobaccili as well as 
bifidobacteria. Hence, the medium was found to be non-specific for 
bifidobacteria . 

YN-17 and MFN were found to be equally selective for bifidobacteria with 
less than 3% contamination by fecal streptococci. Table 2 shows that the two 
media were equal in their ability to recover bifidobacteria from the fecal 
samples. 

MFN required an incubation time of 48h before visible growth occurred, 
exceeding the incubation tinie required for YN-17 by 24h. 

Results of enumeration of bifidobacteria on YN-17 and E^ coli on m-TEC are 
presented in Table 3. Bifidobacterial counts from human feces were found to be 
greater than counts of E^ coli by a factor of approximately 10-^. Table 4 
presents results obtained using HBSA. Sorbitol-fermenting bifidobacteria were 
found in equal densities to those of E^ coli . YN-17 medium supplemented with 
LiCl concentrations of 3% and 4% displayed fecal streptococci contamination 
accounting for < 8% of the total colonies isolated. 

Part 2: Suivival of Bifidohacteria and E. ooli (Table 5) 
Pure Culture of Bifidobacteria Isolated frcm Sewage 

The die-off patterns of bifidobacteria and E^ coli appeared to parallel each 
other (Fig. 2) . However, bifidohsacteria could not be recovered after the 



- 220 - 

TABLE D2 

COMPARISON OF LEVELS OF BIFIDOBACTERLA 

ISOLATED FROM HUMAN FECES ON YN-17 AND MPN 



Bifidobacteria Per 1 Gram 
SAMPLE of Human Feces 



SOURCE 



YN-17 MPN 



Human I 2.1 x 10^ 2.0 x 10^ 



Human A 1.8x10^ 2.0x10^ 



Mean 2.0 x 10^ 2.0 x 10^ 



=LE D3 



- 221 - 

LEVELS OF BIFIDOBACTERIA AND ESCHERICHIA COLI 
IN HUMAN FECES AS ISOLATED ON YN-17 AND M-TEC 



SAMPLE 
SOURCE 



Microorganisms Per Gram of Feces 



Bifidobacterium spp 



Escherichia coli 



Human A 
Human B 
Human C 
Human D 
Human E 
Human F 
Human G 
Human H 



2.3 X 10' 



2.6 X 10' 



1.1 X 10' 



1.2 X 10 



7.9 X 10 



4.1 X 10 



10 



10 



10 



1.4 X 10 



11 



1.8 X 10 



10 



1.8 X 10 



2.0 X 10 



1.6 X 10 



9.4 X 10 



5.0 X 10 



1.6x 10 



3.4 X 10 



2.5 X 10 



Mean 



3.7 X 10 



10 



2.4 X 10 



enumerated on YN-17 



enumerated on M-Tec 



r.^31E Cii 



_ ")2'' - 

LEVELS OF SORBITOL-FERMENTING BIFIDOBACTERIA 

AND ESCHERICfflA COU IN HUMAN FECES AS 

ISOLATED ON HBSA AND M-TEC 



SAMPLE 
SOURCE 



Microorganisms Per Gram of Feces 



Bifidobacterium spp 



Escherichia coli 



.b 



Human A 
Human B 
Human C 
Human D 
Human E 
Human F 
Human G 
Human H 



2.3 X 10' 
1.2 X lo''' 
3.2 X 10^ 
4.0 X 10^ 
2.2 X 10^ 
4.2 X 10'' 
1.9 X 10^ 
3.0 X lO'' 



1.8 X 10' 



2.0 X 10' 



1.6x 10 



9.4 X 10 



5.0 X 10 



1.6 X 10 



3.4 X 10' 



2.5 X 10 



Mean 



2.0 X 10' 



2.3 X 10' 



enumerated on HBSA 



enumerated on m-Tec 



- 11 



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- 225 - 
second day of exposure. E^ coli , in ODntrast, was able to survive as long as 
48h after ejqxDsure to the environmental water, but was not detectable after the 
third day. After the 15h mark the bifidobacteria appeared to decline somewhat 
more rapidly than E^ coli . 
Pure Culture of Bifidobacteria Isolated frcm Human Feces 

The bifidobacteria declined more quicJcly than E^ coli within the first 2h of 
exposure with the bifidobacterial cultures decreasing greater than one order of 
magnitude and the E^ coli cultures decreasing less than one order of magnitude 

(Fig. 3) . Bifidobacterium cultures died off within 24h of exposure to the lake 

water in vitro . 

Pure Culture of Bifidobacterium bifidum (ATOC #696) 

The longevity of Bi f idobacter ium bifidum was relatively consistent with tliat 
observed with the E^ coli culture. However, this pattern appeared to change 
after 5h where the B^ bifidum decreased in numbers much faster than E^ coli . 
After 24h, B^ bifidum could only be isolated from one chamber in low numbers 

(i.e. < l/lmL) . (Fig. 4) . 

Pure Culture of Bifidobacterium breve (ATOC #701) 

Bifidobacterium breve presented a more rapid decrease in density for the 
most part of the survival analysis when compared to E^ coli. Decline of 

B. breve was more marked after 5h of exposure and the species could not be 

recovered after 24h. (Fig. 5) . 

Fecal Sanple 

Both Bifidobacteria and E^ coli appeared to survive maintaining fairly 

steady densities between and 12h (Fig. 5) . However, rapid decline of the 

bifidobacteria occurred after this time interval whereas E^ coli seemed to 

decrease in numbers much more slowly. 















- 225 - 


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- 227 - 



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- 230 - 
DISOJSSICN 

Part 1: Isolation and Enumeraticn 

One of the major problems in assessing an organism's potential role as a 
water quality indicator stems from difficulties in its isolation. Therefore a 
coirparison of the three media: YN-17, MRS, and MPti for their selectability and 
specificity was performed. 

Although visible target colonies were observable on MRS after a relatively 
short incubation time (i.e. 24h) , the medium was not found to be sufficiently 
specific for bifidobacteria. The MRS medium was prepared utilizing the 
antibiotic cocktail devised for YN-17 (Appendix 2) . However, this formulation 
was not able to suppress the occurrence of excessive contamination by 
lactobacilli and fecal streptococci. This occurrence is best explained by the 
fact that the MRS medium was richer in nutrient content than the other two 
media, having been originally developed to support the growth of 
Lactobacillus spp. (Dept. of Nutrition, University of Toronto, personal 
communication) . Although MRS was not appropriate for the specific isolation of 
bifidobacteria, it was utilized (void of antibiotics) , in broth tubes to 
support the growth of pure cultures of these organisms. MRS, as a nutrient 
medium, was found to be suitable for bifidobacteria with visible growth 
appearing as early as 16h after incubation. 

YN-17 and MFN appeared to be equally selective for bifidobacteria as seen in 
Table 2. The results stated previously show that the two media were also 
comparatively specific for bifidobacteria v^ere background fecal streptococci 
were found in levels no higher than 5% on either media. However, as it was 
earlier stated, a potential water quality indicator should possess a relatively 
rapid means of detection and enumeration (Bonde, 1978) . In this regard, YN-17 



- 231 - 
was superior to MPN in that YN-17 required a 24h shorter incubation period in 
comparison to MFN. 

Fecal streptococci were found to be the most successful competitor to 
bifidobacteria where on occasion, the former would overgrow the latter on YN- 
17. Hence a means to better suppress the grcwth of fecal streptococci on this 
medium was sought. Attempts to suppress fecal streptococci contamination using 
0.3% and 0.4% LiCl in YN-17 failed. Fecal streptococci contamination prevailed 
and were found in numbers consistent with those obtained prior to the 
incorporation of LiCl. 

The use of E^ coli as a water quality indicator of recent fecal 
contamination (Seyfried et al. . unpublished report) provides a standard with 
which to compare bifidobacteria. For the reasons mentioned above, YN-17 was 
the mediijm of choice for the enumeration of bifidobacteria from feces and 
subsequent density level comparisons with E^ coli. Also employed was Mara and 
Oragui's (1983) HBSA medium for the enumeration of sortoitol-fermenting 
bifidobacteria. 

The mean density levels of bifidobacteria and E^ coli found in the feces of 
eight human adults have been tabulated (Table 3) . Similarly, the density 
levels of sorbitol-fermenting bifidobacteria in comparison with E^ coli have 
been presented in Table 4. The results of this study showed bifidobacteria to 
exist in feces at a 1000-fold higher concentration than E^ coli. The sorbitol- 
fermenting species of bifidobacteria presented themselves in numbers coitparable 
to those of Ei coli (i.e. 10^) in the sample tested. These values were 
consistent with the bacterial content, found in human feces, reported by Mara 
and Oragui (1983). i 



- 232 - 
Part 2: Survival Study 

Figures 2-6 represent the results obtained form the survival study. In 
every case both bifidobacteria and E^ coli were found to decline after 
exposure to Lake Ontario water, in vitro . Possible reasons for the observed 
die-off include: nutrient depletion, diffusion of nutrients out of the dialysis 
chambers, toxic chemical diffusion into the chamber, and where bifidobacteria 
is concerned, the effects of atmospheric oxygen levels toxic to the anaerobes. 

Pure cultures of bifidobacteria appeared to parallel the die-off trends in 
Ei coli up to the 5h mark; thereafter bifidobacteria began to show a slightly 
more rapid decline (Figures 2, 4, 5). In all cases bifidobacteria died off 
within 24h of exposure whereas E. coli persisted as long as 48h. 

As was noted, the bifidobacteria survived was comparable with E^ coli within 
the first 5h of exposure. It is suggested that, upon exposure to the 
environment, some of the bifidobacteria were able to utilize nutrients from 
dead cells to maintain their existence. Eventually as time passed the effects 
of diffusion, resulting in dilution of nutrients and ejqx)sure of the pure 
culture to any toxic chemicals present in the water, may have had an inpact on 
the survival of the organisms. Also, it is expected that competition for 
nutrients increased over time due to the depletion of nutrients by the 
organisms themselves. The more rapid decline of bifidobacteria in comparison 
to Ei coli may possibly be accounted for by considering oxygen diffusion into 
the chamber. Initially the pure cultures were suspended in autoclaved 
environmental water (see Methods and Materials) . Heating of the water would 
have led to a subsequent driving-off of oxygen. Once the chambers containing 
the suspension of bifidobacteria were placed into the lake water, oxygen 
content may have increased due to diffusion. Thus it was perhaps the toxic 



- 233 - 

levels of oxygen after the 5h mark that lead to the more rapid decline of the 
bifidobacteria at this point. 

Figure 3 presents bifidobacteria and E^ coli in fairly steady decline with 
both cultures becoming undetectable within 24h. The more rapid decline of both 
organisms in comparison to those cultures presented in Figures 2, 4 and 5, may 
have been the result of differences in environmental water utilized in the 
study. Table 1 shows that the water used for the cultures (Fig. 3) was 
retrieved from a boat dock. It is quite possible that this water contained 
residual chemicals, emitted by boat engines, that would have a toxic effect on 
the bacterial cultures. 

Results of exposure of a mixed culture of bifidobacteria are presented in 
Table 6. In this instance, a diluted fecal sample was introduced into the 
dialysis chamber in an effort to present bifidobacteria and E^ coli in vitro , 
as they might be found in the natural environment. 

As can be seen in Figure 5, both organisms tended to exist in a relatively 
stationary state over a 12h period. These results are inconsistent with the 
survival data found using the pure cultures. However, it would be plausible to 
state that the greater nutrient level provided by the diluted fecal material 
within the chamber, allowed the organisms to maintain their numbers. 

It is possible that coitpetition with other organisms found in human feces, 
nutrient depletion, and effects of toxic chemicals resulted in the decline of 
the bifidobacteria and E^ coli after 12h exposure to the environment. 

Once again the more rapid decline phase of bifidobacteria is suggested to 
have been due to the fact that the organisms are strict anaerobes. 



- 234 - 
OCNCmSICNS 

Coiiparisons with E^ coli , presently being used as a water quality indicator 
of recent fecal pollution (Seyfried et al. , unpublished report) , support the 
assumption that sorbitol-fermenting species of bifidobacteria may serve as 
indicators of recent fecal contamination in surface waters. As was shown in 
this study, their densities in feces, their ability to survive in vitro for as 
long as 24 hours, and the existence of rtvethods allowing for their selection and 
recovery si:ipport this proclamation. 

Unfortunately an in vitro survival study poses some limitations. The 
effects of ultraviolet radiation, temperature changes, drying, predation, 
competition, water flow, and pH changes occurring in the natural environment 
could never be mimicked completely in vitro . 

Nonetheless, the fact that certain species of bifidobacteria have been found 
to be restricted to human feces, and their tendency to die off quite rapidly 
make these organisms promising water quality indicators for determining 
pollution sources and locations. 

REOCMMENEftnCNS 

1. A study should be performed in Ontario surface waters to characterize the 
survival trends of bifidobacteria in situ. 

2. An epidemiological study should be performed in Ontario to confirm the 
statements that sorbitol-fermenting species of bifidobacteria are restricted 
to humans. 



- 235 - 

APraMDZX 1 

Riosphate Soluticn/Diluticn Blanks: 

a) Dissolve 34. Og KH2PO4 in SOOmL dH20. Adjust pH to 7.2. Dilute to IL 
with dH2) . 

b) Dissolve 50g MgS04 in IL dH20. 

Magnesium Chloride Soluticn: 

MgCl2 38. Og 

Distilled water (dH20) l.OL 

Stir ingredients to dissolve. Autoclave for 15min. at 121° C (15 lbs 
pressure) . 

Diluticai Blanks: 

Phosphate Solution (1) 1.25mL 
MgCl Solution (2) 5.0inL 

Mix solutions and dispense 99mL (plus 4inL for evaporation) into dilution 
blanks. Autoclave for 15min. at 121° C (ISlbs pressure) . 

Gelatin Riosphate Buffer: 

gelatin 2g 

Na2HP04 3.7g 

NaH2P04 7 . 25g 

dH20 l.OL 

Dissolve in SOOmL of distilled water. Adjust pH to 7.2 ±0.2 with l.ON 
NaOH. Dissolve gelatin in SOOmL of distilled water (heat to < 90° C) . 
Autoclave for ISmin. at 121° C (llSlbs pressure) . 

Urease Reagent: 

Urea 10 . Og 

Ehenol Red O.OSg 

Ethanol . 5mL 

Mix ingredients and adjust pH to 5.0+0.2. 



- 236 - 

AEFENDIX 2 

Growth Media 

1. MRS (oxoid) broth was prepared according to the manufacturer's 
recornmendations . 

2. MRS selective medium was prepared by addition of th YN-17 antibiotic 
cocktail (4a) and 20g of BactoAgar. 



ra-TEC Agar: 




Proteose peptone #3 


S.OOg 


Ye^st extract 


3.00g 


Lactose 


lO.OOg 


NaCl 


7.50g 


K2HPO4 


3.3g 


KH2PO4 


l.OOg 


Sodium lauryl sulphate 


0.20g 


Sodium deoxycholate 


O.lOg 


Bromocresol purple 


0.08g 


Bromocresol red 


0.08g 


Agar 


IS.OOg 


dH-.0 


l.OOL 



Mix above ingredients and heat to 90° C to dissolve. Autoclave for 15min. 
at 121° C (151bs pressure) . Cool to 50° C and dispense in sterile petri 
dishes . Final pH 7 . 1+0 . 1 . 



4a. YN-17 




Yeast extract 


20. Og 


Polypeptone (BBL) 


10. Og 


Lactose 


10. Og 


Casamino acids 


8.0g 


NaCl 


3.2g 


Ecxjmocresol green 


0.3g 


Cysteine hydrochloride 


0.4g 


BactoAgar 


15. Og 


dH20 


IL 


Antibiotics: 




Nalidixic acid 


0.03g 


Kanamycin sulphate 


0.05g 


Polymixin B 


0.006g 



Mix above ingredients with the exception of the antibiotics and heat to 
90° C to dissolve. Autoclave for 15min. at 121° C (151bs pressure) . Cool 
to 50° C and add antibiotics listed above. Dispense in sterile petri 
dishes. Final pH 6.9+0.2. 

b. YN-17 (-): 

Same formulation as above with the exclusion of antibiotics. 

5. Human Bifid Sorbitol Agar (HBSA) : 



- 237 - 



Sorbitol 


10. Og 


Polypeptone (BBL) 


10. Og 


Yea.st extract 


20. Og 


Casamino acids 


8.0g 


NaCl 


3.2g 


Bronocresol purple 


O.lg 


Cysteine hydrochloride 


0.4g 


Bacto Agar 


15. Og 


dH20 l.OL 




Antibiotics: 




Nalidixic acid 


0.03g 


Kanamycin sulphate 


0.05g 


Polymixin B 


0.0012g 



Mix above ingredients with the exception of the antibiotics and hat to 90° 
C to dissolve. Autoclave for 15min. at 121° C (151bs pressure) . Cool to 
50° C and add antibiotics listed above. Final pH 6.8+0.2. 

MFN: 



a) 



b) 



Lactose 






20. Og 


(NH4)2S04 






5.0g 


K2HKJ4 






l.Og 


Twef^n 80 






l.Og 


Bromocresol 


purple 


o.ooieg 


Bacto Agar 






20. Og 


Salt Solution: 


(S.OmL) 




FeS04*7H20 






0.5g 


MnS04*2H20 






0.4g 


MgS04*7H20 






10. Og 


NaCl 






0.3g 


dH20 






250mL 


Biotin 






O.OOOlg 


Pantothenic 


acid 


0.002g 


Riboflavin 






O.OOlg 


Adenine 






O.OOlg 


Guanine 






O.OOlg 


Xanthine 






O.OOlg 


Uracil 






O.OOlg 



Mix ingredients (a) in 740inL dH20 and heat to 90° C to dissolve. 
Autoclave for 15min. at 121° C (151bs pressure) . Cool to 50° C. Mix 
ingredients (b) in lOmL dH20 and sterilize by meiitjrane filtration. Add 
(b) to (a) and adjust pH to 6.8+0.2. 



- 238 - 

REFEEENCES 

1. Bonds, G.J. (1977) Bacterial indicator of water pollution. Adv. Aquatic 
Microbiol. I: 273-364. 

2. Buchanan, R.E. and Gibbons, N.E. (1974) Bergey's Manual of Determinative 
Bacteriology ei^th edn. Williams and WilJcins: Baltiinore. 

3. Cabelli, V.J. (1979) Evaluation of recreational water quality: the EPA 
approach. in BioloqiccLL Indicators of Water Quality . A. James and L. 
Evison (ed.), John Wiley and Sons: Oiichester. pp. 1-23. 

4. Carillo, M. , Estrada, E. and Hazen, T.C. (1985) Survival and enumeration 
of the fecal indicators Bifidobacterium adolescentis and Escherichia coli 
in a tropical rain forest watershed. Amer. Soc. for Microbiol. 5.0(2) : 
468-476. 

5. ■ Dufour, A.P. and Cabelli, V.J. (1975) A membrane filter procedure for 

enumerating the conponent genera of the coliform group in seawater. Appl. 
Microbiol- 29: 826-833. 

6. Dufour, A.P,, Strickland, E.R. and Cabelli, V.J. (1981) Membrane filter 
method for enumerating Escherichia coli . Appli. and Environ. Micrcbiol. 
4195): 1152-1158. 

7. Geldreich, E.E., Best, L.C., Kenner, B.A. and VonDonsel (1968) The 
bacteriological aspects of stormwater pollution. J. WPCF. 40(11): 1861- 
1872. 

8. Huh, I. (1986-87) The isolation and identification of Bifidobacteria. 
University of Toronto, unpublished report. 

9. Levin, M.A. (1977) Bifidobacterium as water quality indicators. in 
Bacterial Indicators/Health Hazards Associated with Water . W.W. Hoadley 
and B.J. Dutka (ed.). ASTM Publications: RvLladelphia . pp. 131-138. 

10. MacDonald, J. (1986) Humber River bacterial sources and pathways study. 
Technical Rf^xDrt #13. A report of the Toronto Area Watershed Management 
Strategy Steering Committee. Prepared for the Ontario Ministry of the 
Environment. 

11. Mara, D.D. and Oragui, J.I. (1983) Sorbitol-fermenting bifidobacteria as 
specific indicators of human faecal pollution. J. Appl. Bact. 55: 349- 
357. 

12. Mossel, D.A.A. (1958) The suitability of bifidobacteria as part of a 
more extended bacterial association indicating fecal contamination of 
foods. Seventh Intematicnal Ocngress of Microbiology Abstracts of 
Papers. Almquist and Wikesells: Uppsala, pp. 440-441. 

13. McFeters, G.A. and Stuart (1972) Survival of fecal coliform bacteria in 
natural waters. Field studies with membrane filter chambers. App. 
Micrcbiol. 24: 805-311. 



- 239 - 

14. Oragui, J.I. (1982) Bacteriological methods for the distinction between 
human and animal fecal pollution. Hi.D. Thesis, University of Cundee, 
Scotland. 

15. Petuely, F. (1956) Ein einfacher vollsynthetischer Selektiv nahrboden fur 
den Lactobacillus bifidus. Zentralbl. Bakeriol . Prasitenkd. 
Infekticnskr. I^. Abt. I. Orig. 166: 95-99. 

16. Resnick, K.G. and Levin, M.A. (1981) Assessment of bifidobacteria as 
indicators of human fecal pollution. Appli. and Evotcti. Micrcibiol- 42: 
433-438. 

17. Seyfried, P et al. (1987) unpublished report. 

18. American Public Health Assoc. (1985) .qharrtaTri Methods for the 
T=^fnmirwl- icn of Water and Wastewater . 16th edn. AFHA, AWWA and WPCF (ed.). 
pp. 886-901. 

19. Tanaka, R. and Mutai, M. (1980) Iitproved medium for selective isolation 
and eniimeration of bifidobacterium. Appl. and Envircn. Micrc±)iol. 40: 
866-869. 



- 240 - 

STECIAL NOTES 

There seenved to exist sane discrepancies between the findings of this 
study and those of Mara and Oragui (1983) in regards to YN-17 medium. Mara 
and Oragui found flat, non-ntucoid colonies measuring 2-4iTim and dark green in 
colour to be background streptococci. Ihey distinguished domed, mucoid 
colonies measuring l-2mm and pale green with a pale green periphery as being 
bifidobacteria. 

However, this study showed the opposite to be true. This is to say the 
smellier dark green/blue flatter colonies were identified as bifidobacteria and 
those ejdiibiting a pale green periphery were contaminating streptococci or more 
likely a mixture of bifidobacteria and streptococci. Any colonies pale green 
in colour that did not contain a darker centre were found to be streptococci. 



- 241 - 



APPDJDIX E 

1 

Clostridiun perfrinqens and Bifidobacterium ^. as Tracers in Storm Sewers 



Eric K. Hani, 
Department of Microbiology 
Iftiiversity of Torcnto 



- 242 - 

TAHTF OF acwTENrs 



Page No. 

LIST OF TABLES ^^^ 

LIST OF FIGURES ^^^ 

245 

METHODS 

Sample Collection ^^-^ 

245 

Analysis 

253 

SUMMARY '^ 



- 243 - 

UBT OF TS^HLES 

Table Page No. 

E-1 Ratio of Ei coli to Clostridiuni perfrinqens 246 

and of Bifidobacteria to Ej_ coli for High 
Priority and Non-Priority Storm and 
Sanitary Sewage 

E-2 Geometric Mean Concentrations of Fecal 247 

Coliforms, E^ coli , Clostridium perfrinqens 
and Bifidobacteria in Hi(^ Priority, 
Non-Priority Storm and Sanitary Sewage 



- 244 - , 

UST OF FIGURES 
Figure Page No. 

E-1 Geometric Mean Concentrations of Fecal 248 

Coliforms, E^ coli . Bifidobacteria spp. and 
Clostridium perfringens in Sanitary Sewage 
during Dry Weather Survey June 10, 11 
and 12, 1987 

E-2 Geometric Mean Concentrations of Fecal 249 

Coliforms, E^. coli . Bifidobacteria spp. and 
Clostridium perfringens in High Priority 
Storm Sewage during Dry Weather Survey 
June 10, 11 and 12, 1987 

E-3 A conparison of the Geometric Mean 250 

Concentrations of E^ coli , Clostridium 
perfringens and Bifidobacteria in Hit^ 
Priority and Non-Priority Storm and 
Sanitary Sewage (DOG values) 

E-4 A comparison of the Geometric Mean 251 

Concentrations of E^ coli , Clostridium 
perfringens and Bifidobacteria in Human, 
Cat and Dog Feces (LOG values) 

E-5 Percentage of Sorbitol Fermenting 252 

Bifidobacteria in Feces - High Priority 
and Non-Priority Storm and Sanitary Sewage 



- 245 - 
METHODS 
Sanple Collecticn 

1. High priority storm sewage- triplicate sanples from three in-line sample 
points. 

2. Sanitary sewage-triplicate saitples fron three in-line saitple points. 

3. Non-priority storm sewage-triplicate saitples from two in-line sample 
points. 

4. Fecal samples from humans, cats, dogs. 
Analysis 

Membrane filtration for parameters: 

Fecal coliforms/E. coli 

m-TEC + urease treatment 
44.5 ± 0.5° C; 23 + 1 hr 
(Dufour, Strickland and Cabelli, 1975) 

Clostridium perfringens 

Heat treatment at 70° C for 30 minutes 

mCP-2 at 37° C for 48 hr 

(Ontario Ministry of the Environment, 1986) 

Bifidcbacteriim sp. 

YN17 at 37° C for 48 hr, anaerobic gas pack, anaerobic jar 

(Mara and Oragni, 1983) 

isolates tested in sorbitol broth) 



- 246 - 



Table E-1 



Ratio of E. Ooli to Clostriditnn perfrinqens and of Bif idcbacteria to 
E- CDli for Hi^ Priarity and Non-Priority Stonn and Sanitary Sewage 



Sewer Sanple 



HP Storm A 
HP Storm B 
HP Storm 
HP Storm 
NP Storm 
NP Storm 
Sanitary 
Sanitary 
Sanitary 



48 

141 
5. 
7. 
7. 

83 

50 

150 



E/C = Ei Coli ; Clostridium perfringens 



Seuec Sanple 


B/E 


HP Storm A 


1.7 


HP Storm B 


2.3 


HP Storm Y 


1.9 


HP Storm C 


0.25 


NP Storm X 


0.01 


NP Storm Z 


0.04 


Sanitary D 


0.02 


Sanitary E 


0.02 


Sanitary F 


0.05 


B/E = Bifidobact 


:erium/E. Coli 



- 247 



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- 249 



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- 253 - 
SUMMARY 
Clostridium perfrinqens 

present in feces in hi^ density 
gcxxi survival ciiaracteristics 
no other source in the environment 
expensive to recover 

no human/non-human determination possible 
indication of age of pollution 
indication of water treatment performance 
Bifidobacterium spp. 

indication of recent pollution 
human/non-human determination possible 
no other source in the environment 
present in feces in high concentration 
no replication extraintestinally 
easy to identify 



- 254 - 



AFFQnnx F 



CHRRftCIHaZftTICN OF PEEDEOCNAS AOHGINDSA 



FRO* STC»M AND SANITRRY SEWERS 



KCm. HABMANDAYAN 

EEPARIMEOT OF MICRDBIOIJDGY, 
UNTVERSnY OF "KMINID 



- 255 - 

TABI£ OF OCNIOnS 

Page No. 

I. INTRDCUCnON 258 

II. OBJECTIVES OF RESEARCH 260 

III. MMERIAIS AND METHODS 260 

Sanple Collection 260 

Enumeration of Pollution Indicator Organisms 260 

Isolation of Pseudomonas aeruginosa 261 

Biochemical Tests 261 

Serotyping 264 

Genotyping (REA) 264 

IV. RESULTS 

Enuineration of Fecal Indicator Bacteria in Sewers. 266 

Storm Sewers 266 

Sanitary Sewers 268 

Serotyping 268 

Genotyping . 272 

V. DISCUSSION 276 

Enumeration of Fecal Indicator Bacteria 

in Sewers 276 

Serotyping 273 

Genotyping 283 

VI. CONCUJSION ■ 286 

VII. APPENDIX 288 
Buffers and Solutions . 288 
Growth Media 289 

VIII. BIBLIOGRAPHY 292 



- 256 - 
UST OF IS^ELES 

Table ' Page No. 

F-1 Media and Incubation Parameters for Enumerating Selected 262 

Bacterial Grotps 

F-2 Serotype Distribution of Pseudomonas aeruginosa in Storm 271 

and Sanitary Sewage 

F-3 Result of Agarose Gel Electrojiioresis of Total Cellular DMA 274 

Extracted From Pseudomonas aeruginosa 

F-4 Correlation of Various Serogroup Typing Schemes Based on Ag 280 

F-5 Worldwide Frequency of Incidence of Serotypes of 282 

Pseudomonas aeruginosa 



- 257 - 

LIST OF FIGURES 

Figure Page No. 

F-1 Gec3metric mean cxjncentration of fecal indicators 267 

in storm sewage 

F-2 Geometric mean concentrations of fecal indicators 269 

in sanitary sewage 

F-3 Percentage serotypes of Pseudomonas aeruginosa 270 

in storm and sanitary sewage 

F-4 Agarose gel electrophoresis of total cellular DNA 273 

from Pseudomonas aeruginosa digested with Sma I 
endonuclease 

F-5 Agaixjse gel electrophoresis of total cellular DNA 275 

from Pseudomonas aeruginosa digested with SMa I 
endonuclease 

F-6 Agarose gel electrophoresis of total cellular CNA 277 

from Pseudomonas aeruginosa digested with SMa I 
endonuclease 

F-7 Agarose gel electrophoresis of total cellular DNA 234 

from Pseudomonas aeruginosa digested with six 
different endonucleases 



- 258 - 

Incnr^ased concern for public hecilth hazard has lead to methods aimed at 
reducing the input of fecal pollution in surface water bodies. Human fecal 
material in sanitary sewage, contains bacteria which may cause infections in 
the appropriate host. Under normal circumstances, this sanitary sewage is 
treated in wastewater treatment plants, reducing bacterial numbers, and the 
effluent is discharged into natural waterways. This process serves to mininiize 
the contact between humans and human fecal material thereby decreasing the 
probability of infection. 

Recently, the Toronto Area Watershed Management (TAKM) has shown that 
storm sewers contribute to the fecal pollution of the Humber and Don Rivers. 
Originally these sewers were designed for channelling storm water from urban 
areas into surface waters to avoid the flooding that would have resulted, as a 
consequence of the bloc3djig of natural flow patterns that existed before 
urbanization. The bacterial content of storm sewers should be similar to 
direct runoff and have virtually no human fecal irpjt. 

High fecal bacterial levels detected in storm sewers suggests that these 
sewers may be contaminated with human fecal wastes. Ihe problems resulting 
from this are obvious: if sewage enters the surface water supplies untreated, 
the probability of infecting a suitable host is high. Therefore, in order to 
determine which home connections are contributing to fecal pollution, some 
method of tracing the pollution must be developed. 

One opportunistic pathogen that has been recovered from human fecal 
material and sewage is Pseudomonas aeruginosa . Ringen and Drake (1952) have 
shown that even though Pseudomonas aeruginosa can be isolated from a wide 
variety of natural sources, its distribution is limited. They found that 



- 259 - 
Pseudomonas aeruginosa was not isolated in natural waters of mountain streams 
or deep wells remote from human habitation and free of human waste material. 
In addition Wheater and coworicers (1978) have shown that Pseudomonas aeruginosa 
was not found in a variety of animal feces. The presence of this organism in 
barnyards, animal feces and soil was due to chance and close association with 
man. Therefore, the presence of Pseudomonas aeruginosa in surface waters and 
storm sewage may indicate the presence of human sanitary wastes. 

Subspeciation of Pseudomonas aeruginosa has classified this microorganism 
into 17 different serotypes according to their somatic-O-antigen. Studies by 
Seyfried and Fraser (1977) and Young and Moody (1974) have shown that tracing 
the Pseudomonas aeruginosa infection in clinical and environmental settings is 
possible. Therefore, by adopting their methods of pollution source tracings in 
storm sewers, one may be able to determine the presence of human sanitary waste 
in storm water and trace the location of the inputs. 

With a limited amount of information on the distribution of serotypes of 
Pseudomonas aeruginosa and their distribution in the environment, work must be 
performed to determine if there is a difference between serotypes of 
Pseudomonas aeruginosa found in humans and those from environmental sources. 
If a serotype, specific to sanitary wastes, if found, then isolating the same 
serotype from storm sewers might indicate the presence of sanitary waste. 

Another method vAiich may prove valuable in determining if a specific type 
of Pseudomonas aeruginosa is associated with human feces, is restriction enzyme 
analysis. This method may be able to determine if a specific Pseudomonas 
aeruginosa genotype is present in human fecal material as compared to 
environmental sources. This in turn may serve as the probe used to determine 
the presence of human Pseudomonas aeruginosa contamination in storm sewers. 



- 260 - 
OBJECTIVES OF RESEARCH ■: 

The objectives of this research were to: 

1. Characterize isolates of Pseudomonas aeruginosa from sanitary and storm 
sewage according to their serotypes and genotypes; in order to see vvtiat 
differences and/or similarities exist among isolates obtained from these 
types of waste and to apply this knowledge to develop a method of detecting 
the presence of sanitary waste in storm sewage. 

2. Etetennine which method (serotyping or genotyping) will give more discrimina- 
ting information about a specific strain of Pseudomonas aeruginosa . 

MATERIALS AND MEIHCCS 

3.1 Sanple Collection 

Sairples were collected from three different points along the storm sewer 
line, in triplicate, over a three day period. 

Samples were also collected at three points along the sanitary sewer 
lines. 

Water samples were collected in sterile Sodium Thiosulphate EDTA treated 
bottles (Appendix) and transported to the laboratory on ice. Upon receipt of 
the samples, analysis was performed within three hours. 

3.2 Enumeration of Pollution Indicator Organisms 

Appropriate dilutions, ranging from 50ml to lO"'^, of each water sample 
were made in phosphate solution (Appendix) . Water samples were analyzed using 
standard membrane filtration technigues (APHA, 1989) . Water samples were 
filtered through 0.45 ^^m porosity membranes (Gelman Sciences) and 



- 261 - 

subsequently placed on selective media. Table 1 lists the media, incubation 
times and temperatures used for quantitation of different bacterial groups. 

3.3 Isolatioi of PseudamoTas aenninosa 

Approximately 10 bacterial colonies were picked form m-PA plates, after 
the incubation period, and streaked onto nutrient agar (Difco) plates for 
isolated colonies. Plates were incubated at 35° C for 24 hours. One isolated 
colony was picked from the nutrient agar plates and incubated at 35° C for 24 
hours. Cultures were maintained on BHI (Difco) slants overlaid with sterile 
paraffin oil. These slants were maintained at 20° C. 

3.4 Bioctiesnical Tests Performed en PseiidnnnrBS aeruginosa 

An inoculum of the culture was removed from the EHI slant and streaked on 
BHI agar plates for two consecutive transfers. Plates were incubated at 35' C 
for 24 hours. 

1. - 3% KDH Test 

The 3% KDH test (Fluharty and Packard, 1967) was used to determine the 
gram reaction of each isolate. A small inoculum of the culture was mixed 
with a drop of 3% KDH solution on a slide. The suspension was observed 
for consistency. 

Gram-positive organisms did not gel when mixed with KOH while gram- 
negative organisms ( Pseudomonas aeruginosa ) form a thick stringy gel. 
Results were recorded immediately. 



- 262 - 



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2. - Oxidase Production 

A 1% solution of N, N, N, N- tetramethyl -p-phenylene diamine (Appendix) 
was used to detect the presence of oxidase. The solution was impregnated 
on clean Whatman No. 5 filter paper discs and allowed to dry. Cultures 
were taken from heart infusion agar plates with sterile toothpicks and 
rubbed into the treated filter disc. 

Cultures possessing oxidase produce a purple colour on the filter paper, 
while the negative cultures remained colourless. Results were recorded 
within two minutes, Pseudomonas aeruginosa is oxidase positive. 

3. - Acetamide Reaction 

Each Pseudomonas isolate was streaked onto acetamide agar slants 
(Appendix) and incubated for 24 to 36 hours at 37° C. The production of 
a dark pink (purple) coloration of the slant by Pseudomonas aeruginosa 
was regarded a positive result. 

4. - Skim Milk Reaction: (Brown and Foster, 1970) 

Skim Milk agar (Appendix) was used to determine four reactions; pigment 
production, casein digestion, fluorescence and grape-like odour by 
Pseudomonas aeruginosa . Pigment was read as green, yellow or brown. 
Casein digestion was evidenced by production of a clear zone in the agar 
around each isolated colony. Cultures were also tested for their ability 
to fluoresce under a Wood's lamp. Pseudomonas aeruginosa cultures were 
found to digest casein, produce a grape-like aroma, have a green-yellow 
pigment, and fluoresce blue-green under UV light. 



- 264 - 

3.5 - Serotyping 

Serotyping Kit (Difco number 3081-32) was used to serotype Pseudomonas 
aeruginosa . Before serotyping, the diluted antisera was tested against its 
specific antigen (Difco number 3082-32) for positive control, also it was 
tested against rabbit serum for negative control. 

A day before serotyping each pure Pseudomonas aeruginosa was streaked out 
on nutrient agar and incubated at 37° C for 18 to 24 hours. 

The day of serotyping, the culture was swabbed with a sterile swab, and 
suspended in 0.85% saline. The suspension was autoclaved for 30 minutes at 
121° C, followed by centrifugation at 1000-2000 rpm for 10 minutes. The 
si^jematant was discarded and the bacterial mass was resuspended in 0.75 ml 
Merthiolate saline (1:10000 Merthiolate in 0.85% NaCl solution) . 

This constitutes the stable heated antigen and it was tested against each 
antisera on slide agglutination. 

3.6 Restriction Enzyme Analysis of PsFaximiCTTas aeruginosa 

Total cellular DMA was extracted as follows: (Bradbury et al, 1984; 
Bradbury et al, 1985) . 

A 1.5ml volume of an 18h nutrient broth culture inoculated with 
Pseudomonas aeruginosa was transferred into a 1.5ml Eppendorff tube and 
centrifuged in a Microfuge 12, Bec3<man for three minutes at 7500 x g. The 
supernatant was discarded and the pellet loosened by vortexing. A 291 ul 
volume of FEB I buffer (Appendix) containing lOmg/ml lysozyme was added and the 
mixture incubated for 20 minutes at 35° C. A 9ul volume of 5M NaCl was added 
and mixed well. A 150ul volume of 10% SCS (Appendix) was added. The solution 
was mixed gently and incubated for ten minutes at 37° C. After 450 >ul volume 



- 265 - 

of 25:24:l:phenol: chloroform: Isoamyl was added to the tube, the mixture was 
vortexed and centrifuged for six minutes at roan temperature at 7500 x g. The 
upper aqueous phase was removed with a pasteur pipet and transferred into a 
l.Sral Eppendorf tube. One ml of 95% cold ethanol was added, the tubes 
vigorously shaken and stored at -20° C overnight. The mixture was centrifuged 
for three minutes at 12000 x g, the supernatant discarded and the pellet 
redissolved in 250 ml Dt^ wash buffer (Appendix) . A total of 500 ul of 95% 
cold ethanol was added, the mixture stored at -20° C for 20 minutes, and 
centrifuged at 1200 x g for three minutes. Ihe supernatant was discarded and 
the pellet allowed to dry at 37° C for 10 minutes. Ihe pellet was dissolved in 
100 ul of distilled water and stored at 4° C until digested. 

Restriction digests were performed using Sma I according to the 
manufacturer's instructions (Boehringer Mannheim) . A 10 ul aliquot of 2x Sma I 
buffer was delivered to an Eppendorf tube and 10 ul of extracted n^ was added. 
A 2 ul sartple of Sma I enzyme was added to the mixture and incubated for one 
hour at 37° C for conplete digestion to occur. A one ml volume of 0.15M CDIA + 
0.4 mg/ml RNase A was added and incubated for 20 minutes at 37° C. A five ul 
volume of 5x sairple buffer was added to the restriction digest. Samples were 
electrophoresed on 0.7% agarose gel for 16 hours at 27 volts. Gels were 
stained with one mg/ml Ethidium bromide in 1 x TAE (Tris base, 1.0 sodium 
acetate, 0.1 M disodium EDIA) for one hour and destained for 3 hours in 
distilled water. Photography was done using UV light at 300nm and a red No.23A 
polaroid 665p/N film with an exposure time of 30 seconds. 



- 266 - 
Results 
4.1 Enumeraticn of Fecal Indicator Bacteria in Sesirers 

Fecal coliform, E^ coll . fecal Streptococci and Pseudonionas aeruginosa 
density were performed on all sanples from storm and sanitary sewers, using the 
membrane filtration technique and media described in Table 1. 

4.1.1 Storm Sewar 

- Fecal coliform count in storm sewer varied from 10-^ - 10** coliform per 
100ml (Fig. 1) . 

Ei coli counts were in the 10-^ E^ coli per 100ml range (Fig. 1) . 

- The level of streptococci was relatively consistent at three points. 

- Pseudomonas aeruginosa levels were lew cotrpared to other bacterial 
indicators. Storm sewers should not contain any human fecal iiput, and 
therefore should have low numbers of Pseudomonas aeruginosa organisms. 
Geldreich and Kenner (1969) proposed that the sources of fecal bacterial 

pollution may be differentiated by using a fecal coliform to fecal streptococci 
FC/FS ratio. It was suggested that if FC/FS ratio is greater than 4.0, the 
source of pollution is likely of human origin; while if the ratio is less than 
0.7 then the source is probably of non-human origin, FC/FS ratios between 0.7 
and 4.0 have been considered as intermediate mixed. FC/FS ratios must be 
eirployed with some degree of caution as it is time dependent; once discharged 
into receiving waters, the differential dieoff rates of these organisms and 
diverse environmental factors may alter their interrelationship to such an 
extent as to render the FC/FS ratio of limited or no significance in 
determining the source of bacterial contamination. 



- 26- 



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- 268 - 
The three sites contained very high counts of fecal col i forms. Usually, 
storm sewers contain little feccLL contamination, however at this time it is not 
known v*iat kind of contamination is contributing to the counts observed. 

4.1.1 Sanitary Sewer 

Sanitary sewers usually contain human fecal waste material ^ich is 
treated before being deposited in water bodies. 

Fecal coliform, E^ coli . feccil streptococci concentrations were almost 10 
fold more than the levels observed in storm sewers (Fig. 2) . 

The Pseudomonas aeruginosa count was observed to be around 10000 per 
100ml in the sanitary sewers. This was considerably hi^er than the counts 
observed in storm sewers. 

4.2 Serotyping 

In total 285 isolates were serotyped; 148 isolates from storm sewers and 
137 isolates from sanitary sewers. The distribution of Pseudomonas aeruginosa 
serotypes in storm and sanitary sewage varied (Fig. 3 and Table 2) . 

In storm sewers (Table 2), the most prevalent serotype was 0:6, about 
72.3%; followed by serotypes 0:1(15.5%), 0:16(5.4%), 0:11(4.7%), 0:8(1.1%) and 
0:12(0.7%). 

Sanitary sewers also had serotype 0:6 as the predominant serotype 
(56.2%). Other serotypes observed from sanitary sewers were serotype 
0:10(21.9%), 0:11(13.1%), 0:1(4.4%) and serotype 0:2, 0:4, 0:5, 0:7 were 
present in very low levels (1.5%) . 

Serotype 0:16 only appeared in storm sewers, v^ereas serotype 0:10 was 
found only in sanitary sewers; however, the percentage recovered was not as 
high as serotype 0:6. 



- 269 - 



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- 271 - 
Taole F-2 






Total nuncar of isolataa : 2S5 



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- 272 - 
4.3 Genotyping 

Genotyping was carried out according to the Bradbury et al . 1984, 
Bradbury et al . 1985 method. Isolates randomly chosen with different serotypes 
were subjected to restriction enzyme analysis. 

Figure 4: - 

Lane 1 contained lambda bacteriophage marker digested 
with Hind III. 

Lane 2-8 contained isolates from storm sewers with 
serotype 0:5. 

Lane 9 and 10 contained isolates from sanitary sewers 
with serotype 0:6. 

Examining Figure 4, it is possible that the same serotype from storm or 
sanitary sewers have different genotypes (restriction pattern) . 

Lanes 2, 3, 4, 7 and 8 have the same restriction pattern whereas lanes 5 
and 6 have different banding patterns from the previous ones. 

Lanes 9 and 10 are from sanitary sewers, serotype 0:6, but each has a 
different restriction pattern compared to the other isolates. 

Four different patterns with genotype 0:6 were observed (Fig. 4, Table 

3) : Two different patterns from storm and two different patterns from sanitary 

sewers. 

Figure 5: 

lanes 2 to 6 contain isolates from sanitary sewers with 
serotype 0:6. 

Lanes 7 and 8 contain isolates from sanitary sewers 
with serotype 0:1. 

Lanes 9 and 10 contain isolates from storm severs with 
serotype 0:1 Not all isolates with serotype 0:6 had 
the same genotype (Fig. 5), lanes 2, 3, 5 and 6 were 
identical to each other, whereas lane 4 had a totally 
different banding pattern. 



_ T7-) _ 



Lan9 
Source 

Serotyp* 
RZA Pattam 

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- 274 - 

Table ?-3 
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DNA 3L':TR.\CTi:D FHCM PSZUDOI.IONAS .^RUGIZtOSA R-^'iDOr.!,'/ : 



JX'J 



ISCLATS NuT.IBZR SII£ SEROTYPE 1--.::Z RZ^ FATTEr.:; 



PXG '4 332 storm A 6 2 

^00 storr: 563 

^20 storr. 36^ A 

1^55 stom A 6 7 --- 

1^90 zzoirz 3 6 S 

T'Z S 1235 sani^sr;/ ? 5 2 

13^5 3arJ.-ary ? 6 2- 

13^0 ss-ii-ary Z c 5 

1570 sarJ-tary Z 6 5 .- 

— T^ u o"! ' o — -,v~- J. i< r - 

rij H :^— ^ o«a-.u .- ^ ^ — 

9^0 sxorr: 3 6 6 ' 2 

53s sajiitary F 6 9 C 

1505 sanitar:.- D 6 10 D 

I52B sanitazy D 6 iJ- Z 

-I527 
1530 

^{-21 

i^36 



FIG 6 361 

393 



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3 : sanitary 



- 276 - 

Lanes 7 and 8 had the same restriction pattern and both 
were from sanitary sewers serotype 0:1; however, when 
these were coirpared to lane 9 and 10 they displayed a 
different restriction pattern. Serotype 0:1 from 
sanitary sewer had a different fingerprint from storm 
sewer isolates with serotype 0:1. 

Coitparing Figure 4 to Figure 5, seme serotype 0:6 
organisms from storm sewer had an identical pattern 
or serotype (Table 3) , to sanitary sewer serotype 
0:6. Therefore, in this case, serotype 0:6 from 
storm sewer had two different genotypes whereas the 
sanitary sewer had four different genotypes. 



Figure 6 



Lanes 2 and 3 had isolates from storm severs with 
serotype 0:16; both had identical patterns whereas 
lanes 4 and 5 contained isolates from sanitary sewer 
serotype 0:10 with totally different genotype each. 

Lane 6 contained an isolate fron sanitary sewer with 
serotype 0:11 which had different pattern compared to 
serotype 0:11 in lanes 8 and 9 from storm sewers. 

Lanes 7 and 10 each contained different serotypes, 0:4 
from sanitary sewer and 0:8 from storm sewer, they 
had totally distinct restriction pattern from each 
other. 



DISCUSSICN 

5.1 Enumsratlcn of Fecal Indicator Bacteria 

The result of the study (Fig. 1 and Fig. 2) show that Pseudomonas 
aeruginosa was found in high concentrations in the human sanitary sewers in 
relation to high fecal indicator counts. Pseudomonas aeruginosa was also 
recovered in the storm sewers although in Icwer concentration than in the 
sanitary sewer. The concentration in storm sewer of both fecal col i form and E. 
coli was high especially at point A and approached levels of 10,000 
organisms/ 100ml. FC and EC concentration of this magnitude are suspiciously 
high for storm water and suggest the presence of sanitary contamination or an 
extra input from somewhere else. At this point conclusions can not he irade 
about the source of the contamination. 



- 277 - 



Lana 

Source 

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cellular DNA from ?S£U0CKONAS AERUGINOSA 
digaatid with S.Tia I ^ndonuclsaaa . 



s : jtorm 
3 t sanitary 



- 278 - 
5.2 Serotyping 

The cxsncept of serological heterogeneity of Pseudonpnas aeruginosa was 
first recognized in 1912 by Jacob Sthal, who reported that the 55 strains he 
was investigating were not serologically hcmogenecus (Verdes and Evans, 1961) , 
The heterogeneous nature of this bacterium was also noted in 1916 by 
Trommsdorff , who emphasized the iirportance of distinguishing between the 
different types of Pseudomonas aeruginosa (Verder & Evans, 19161) . These 
authors failed, however, to recognize and differentiate between the heat stable 
(0) and heat labile (H) antigenic ccaiponents of Pseudomonas aeruginosa . The 
presence of two different antigens was first r^xDrted by Brutsaert (1924) and 
the different antigenic conponents were investigated specially (Monoz et al . 
1945, 1949; Gaby, 1946; Mayr-Harting, 1948; Fox and Loutoury, 1953; L^wbury and 
Fox, 1954) ; however, the develcpnent of a practical typing scheme, based on the 
antigens of Pseudomonas aeruginosa , was slow. 

Habs in 1957 used boiled antigens and immune sera prepared against these 
suspensions to devise the first antigenic scheme, suitable for the practical 
differentiation of Pseudcanonas aeruginosa . She reported the differentiation of 
70 strains of Pseudomonas aeruginosa into 12 serogroups, based on heat stable 
agglutinogens. Cross reactions between grtxps 0:2 and 0:5 were evident and 
there appeared to be a common antigen among all the strains. Kleinmaier (1957) 
confirmed Habs' work using slide agglutination with living suspensions of 
Pseudomonas aeruginosa . 

SandviJc (1960) developed an antigenic scheme for this bacterium 
containing seven groups, into which he could subdivide 87 strains of 
Pseudomonas aeruginosa of animal origin. When it was compared with Habs' 6/7 
serogroups were the same, but the uncommon serogroup was added to the Habs' 
scheme as 0:13. Veron (1961) analyzed Habs' cross reacting serogroups 0:2 and 



- 279 - 
0:5 and subdivided 0:2 into 0:2a and 0:2b and 0:5 into 0:5c and 0:5d. Veron 
also emphasized the necessity of cross absorption in order to eliminate the 
cross reactivity and produce monospecific typing serum thus Veron described 10 
O-groups and 10 H-grot^s. 

lanyi (1966/67) further extended the work of Verder & Evans (1961) ; he 
reported 13 types of whicii 5 would be further divided. 

Fisher et al. (1969) described a new concept in serotyping of Pseudonxpnas 
aeruginosa . Tliey differentiated strains of this bacterium on the basis of 
protective antigens and reported on immunotypes. 

Homma et al. (1970) developed a typing scheme, based on her 0-serotypes, 
tested by tube agglutination. 

Bergan (1973) coipared the typing sets of Habs, Lanyi and Sandvik and 
reported as the others had, that they were similar. Since then an 
international panel under the auspices of the subcommittee on Pseudomonas 
aeruginosa and related organisms came v^) with 17 different serotypes. 

Types 1 -12 were pr^ared using Habs' culture 

Type 13 with Veron 0:13 (Sandvik's type 11) 

Type 14 with Verder and Evans' 5 

Type 15 with Lanyi 's 12 

Type 16 with Homma 's 13 

Type 17 with Meitert's type X strain 

Table 4 shows the correlation of various serogroups typing schemes based 
on antigens up to 1969. 

Serotyping of Pseudomonas aeruginosa was carried out using " Pseudomonas 
Antisera " Kit (Difco) which utilizes the heat stable 0-antigen. Of the 285 
isolates serotyped, 148 were isolated from storm sewers, and the remaining 137 
from sanitary sewers. 



280 - 



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- 281 - 

Table 2 lists the distribution of serotypes of Pseudomonas aeruginosa in 
storm and sanitary sewage. Serotype 0:6 was the most frequently identified 
serotype in storm and sanitary sewers, 72.3% and 56.2% respectively. Lanyi 
(1966, 1967) has also r^xsrted that the same serotype was the most common 
serotype isolated from sewage and surface waters. 

Serotypes 0:1 and 0:11 were commonly isolated from storm and sanitary 
sewers (Table 2) . These serotypes were also frequently encountered in studies 
by Muraschi and coworkers (1966) , Lanyi (1966/67) ; Table 5 shows worldwide 
frequency. 

Serotype 0:16 was only recovered from storm sewers, with a frequency of 
5.4% and serotype 0:10 recovered frcxn sanitary sewers 21.9%. This suggests 
that serotypes 0:16 and 0:10 each belong to a specific source. In theory, 
storm sewers should only have direct run off from streets, domestic animals, 
and birds' fecal contamination and sanitary sewers should have human waste 
inputs. Therefore, since type 0:10 was found in a high frequency in sanitary 
sewers, and was absent from storm sewers, this serotype may be specific and 
indicate human fecal contamination. However, at the present time it is not 
known if serotype 0:10 is specific to humans. Sampling of fecal material from 
domestic and wild animals should be performed in order to determine if serotype 
0:10 is found in animal wastes and if so, at v^^iat frequency? 

If serotype 0;10 was found to be specific to human feces, water samples 
of storm sewers taJcen from various points could be performed and tested for the 
presence of Pseudomonas aeruginosa serotype 0:10. If the sample was positive 
for serotype 10, then the sampling site could be examined and the source of the 
illegal connection determined. 

Serotyping by itself does not differentiate within a specific serotype. 
E'/en though serotypes 0:1, 0:6 and 0:11 were common to both storm and sanitary 



- 282 - 






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- 283 - 
sewers, this does not mean that they are identical. To ascertain if this was 
the case, restriction enzyme analysis was performed on serotype groups in order 
to determine if identical genotype patterns are present within each serotype 
groi^j. 

5.3 Genotyping (REA) 

Initially REA was performed on three isolates using six different 
restriction enzymes. Ihis step was used to determine which restriction enzyme 
cut the genomic CNA and produced the best banding patterns on polyacrylamide 
gels. Figure 7 displays the different banding patterns that were obtained. 
Sma I produced the best pattern because hi(^ molecular weights of CNA bands 
were distinct and separate unlike the bands produced by Hind III, Bam Hi, Eco 
RI, KFN I and Xho I. Furthermore Sma I is six base cutter cuts at GGGCCC 
points since Pseudomonas aeruginosa has high G/C content of 67%. This 
indicates that the hi<^er G/C content of Pseudomonas aeruginosa may make it 
more susceptible to cutting by Sma I. 

Twenty-seven isolates of Pseudcanonas aeruginosa were chosen from storm 
and sanitary sewage and genotyped. Isolates were chosen randomly according to 
their serotype. 

Figure 4 displays the genotypes of sewer isolates from storm sewers, 
serotype 0:6 and two isolates from sanitary sewers, serotype 0:6. The banding 
pattern of the storm sewer isolates Figure 4 lanes 5 and 6 were not consistent 
with others. Isolates in lanes, 2, 3, 4 and 5 are identical where those in 
lanes 9 and 10 differ. In comparison, five isolates from sanitary sewers, 
serotype 0:6 Figure 5, lanes 2 -6 did not display the same genotype pattern. 
Isolates in lanes 2, 3, 5 and 6 were identical, while the isolate in lane 4 was 
different. 



- 234 - 







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Identical genotype patterns were observed using serotype 0:16 (Figure 6 
lanes 2 and 3). Serotype 0:1 fron storm Figure 5, lanes 9 and 10 were 
identical whereas serotype 0:1 from sanitary Figure 5 lanes 7 and 3 were 
identical but different frcm storm. 

Different genotype patterns were observed within serotype 0:11 from storm 
sewer Figure 6 lanes 8 and 9 and serotype 0;10 Figure 6 lanes 4 and 5. 
Differences within the specific genotype were noted in the high molecular 
weight positions, usually at MW 23.13 K daltons. 

Differences were also noted when coraparing serotype 0:6 sanitary Figure 4 
lanes 9 and 10, and serotype 0:6 storm Figure 4 lanes 2-8. 

Comparing Figure 4 and Figure 5, it is possible to conclude that sane 
serotypes 6 from storm and sanitciry sewer are identical (i.e. they have the 
same genotype Table 3) , but at this tiite one cannot make any conclusion about 
the presence and the source of this genotype in storm sewers. 

The differences that were seen in REA were not observed using the 
serotyping technique. For example, not all serotype 0:6 isolates possessed the 
same genome pattern. This may be accounted for by the fact that the genes 
responsible for serotyping the 0-Ag canprise a small percentage of the total 
genome. Therefore, when comparing total chromosamcLL CNA other genes besides 
those that code for the 0-Ag, which is characteristic of serotype 
differentiation, will appecir. Thus, the technique of REA proves to be highly 
specific, in that it further subdivides one serotype into a branch of different 
genotypes whic±i can then be used in tracing bacteriail contaminants from 
sanitary sewers into storm sewers. 

It should be noted that genotypes of Pseudomonas aeruginosa isolates have 
not been reported by other researchers. 



- 286 - 

Serotyping, used by itself, was insufficiently discriminatory and of 
little ^idemiological value as some serotypes, namely 0:1, 0:6 and 0:11 were 
cammonly encountered. Conversely, genotyping produced highly specific bands 
and differences within strains of Pseudcno nag aeruginosa were readily 
demonstrated. 

It has been previously suggested that serotype 0:10 '^as e^/ident only in 
sanitary sewers. In addition, the genotype banding patterns were observed to 
be different (Figure 5 lanes 4 and 5). If serotype 0:10 was found in low 
frequencies in other animal groups, REA may provide highly discriminatory 
genotypes specific to humans that can be used in tracing the source of input of 
Pseudomonas aeruginosa in storm sewers Iron a specific site. 

Future studies using a probe developed from either total chramosomal CNA 
or a specific fragment common to human strains will allow us to hybridize 
against cLLl other strains. 

Ihis information would prove valuable in differentiating between human 
and non-human fecal waste. 

(XNCmSICN 

Some common serotypes of Pseudomonas aeruginosa are present in both 
sanitary and storm sewers. The most frequently occurring serotype was 0:6. 

Serotype 0:10 appears to exist only in sanitary sewers whereas serotype 
0:16 was found only in storm sewage. However no definite conclusion can be 
made at this time as to the origin of these serotypes (i.e. sanitary or non- 
sanitary contamination) without further study of the Pseudomonas aer'jginosa 
serotypes found in human and animal feces and those recovered from non-fecally 
polluted environments. 



- 287 - 

Genotyping is more discriminative tJian serotyping, since it can highlight 
both differences and similarities within a given serotype. 

This suggests that REA could be used to specially identify strains from 
sanitary wastes in storm sewage. 



- 288 - 
APPEMUX 
7.1 Buffers and Soluticns 

1. Sodivm Ttiinpailphate/UJIA: 

^2^2*^3 " ^2^ 2-^*? 

EDIA 37 . 2g 

Distilled water (dH20) 100. 0ml 

Stir ingredients to dissolve. Add 0.3ml of buffer to each 120ml sample 
bottle. Autoclave 20 min. at 121° C (15 lbs. pressure) . 

2. Efiosphate Soluticxi: 

(A) Dissolve 34. Og KH2PO4 in 500ml dH20. 
Adjust pH to 7.2. 

Dilute to 1 liter with dH20. 

(B) Dissolve 50g y!gSO^ . 7H2O in IL dH20. 

Autoclave both solutions sepeirately for 15 minutes at 121° C. Cool ard 
store at 4° C for up to 1 month. Add 1.25ml of (A) ard 5ml of (B) to IL 
of dH20. Dispense as dilution blanks or for rinse water. Autoclave for 
20 minutes at 121° C. (15 lbs. pressure) . 

3. Saline (0.85%) 

Add 8.5g of NaCl per litre of dH20. Dispense 5ml in each test tube. 
Autoclave 15 minutes at 121° C. Store at 4° C. 

4. FEB I 

SOnM Glucose, lOmM (EDIA), 25iiiM Tris HCL (FH 8.0) 
lOmg/ml lysozyme 

5. CNA Wash Buffer 

0.1 M sodium acetate, 50mM ^DPS FH(8.0) 

6. 5 X Sample Buffer 

1.0ml of 50 X TAE 

2.5ml of 1% Brtarcphenol Blue in 50% ethanol 

7. 50 X TAE 

302. 5g Tris base, 136. 08g sodium acetate 
tri-hydrate (or 82.03g of anhidrous) 
37. 5g Na2EEnA . 2H2O 



- 289 - 

7.2 Growth Media 

The following media (Difco) was pr^ared according to manufacturer's 
reccamnendation : 

1. ' Nutrient agar 1.5% 

2. Nutrient broth 

3. EHI agar 

4. Skim MiUc Agar: 

SJdjn miUc power lOOg 
Agcu: 15g 

dH20 LL 

Add skim milk powder to 500ml dH20. Stir without heat for 30 
minutes. Add agar to 500ml dH2 and heat to dissolve. 

Autoclave solutions separately for 12 minutes at 121' C. Cool solutions 
to 55' C. Add milk to agar solution aseptically. Mix thoroughly and 
dispense into plates. Final pH 6.4 +/- 0-2 . Store at 4H C. 

5. Medium for the Isolaticn of ttv^ttto Tolerant E. ooli 

(m-TEC - Mug agar) : 

Proteose peptone No. 3 10. Og 

Yeast extract 4.0g 

Lactose 5 . Og 

NaCl 7.5g 

KH2HPO4 3 . 3g 

KH2PO4 l.Og 

Sodium Lauryl Sulphate 0.2g 

Sodium deoxycholate O.lg 

Agar 15. Og 

MDG 0.05g. 

( 4-inethylumbellif eryl-B-D-glucuronide) 

dH20 IL 

Mix above ingredients except MDG to IL dH20 and heat to 90' C to 
dissolve. Add MUG just beforn autoclaving. Autoclave for 15 minutes 
at 121' C. Cool to 55-60' C and dispense in sterile square petri 
dishes. FL'-al pH 7.1 */- 0.1. Store at 4' C. 



- 290 - 

Agar (mENT) : 

M-Enterococcus agar (Difco) 42g 

Sterile dH20 IL 

Wei(^t out agar in a sterile beaker using an alcohol flamed spatula. 
Heat to dissolve agar (93° C) . Cool and dispense into square petri 
dishes. Final pH 7.2 +/- 0.2. 

Medium for Pspiiimmas aeruginosa (m-PA) : 

L-lysine monohydrochoride 5.0g 

Yeast extract 2.0g 

Xylose 2.5g 

Sodium thiosulfate 5.0g 

Magnesium Sulphate, anhydrous 1.5g 

Sucrose 1.25g 

lactose 1.25g 

Sodium C2iLoride 5.0g 

Ferri ammonium citrate 0.30g 

Sodium disoxycholate O.lOg 

Hienol red O.OSg 

dH20 sterile 800ml 

Mix above ingredients and adjxjst pH to 7.6. Add 15g agar. Heat to 
93° C to dissolve agar. Cool to 60° C and stir in Antibiotic 
solution*. Dispense into square sterile petri dishes. Store at 4° C 
after solidification. Final pH 7.1 +/- 0.1. 

* Antibiotic solution: 

A) Sulfapyridine 0.1760g 

B) Kanamycin sulphate 0.0085g 

C) Naladixic acid 0.0370g 

D) Cycloheximide . 1500g 

Dissolve A-D in 200ml sterile dH20. Heat to 50° C to dissolve 
antibiotics. 



- 291 - 

Acetamids Agar Slants 

NaCl 5g 

K2HPO4 1.4g 

KH2PO4 0.7g 

Acetamide 10. Og 

I^ SO4-7 H2O 1-Og 

Ehenol red 0.012g 

Agar 15. Og 

dH20 U;. 

Add all ingredients except agar. Mjx:st pH to 6.8 then add agar. 
Heat to 92' C, autoclave for 15 minutes at 121' C. Final pH 6.3 V 

0.2. Dispense into sterile screw Ccip test tubes. 



- 292 - 

RTnr.TOGRftFHY 

Berrgan, T. 1973. Epidemiology Mar]cers for Pseudcmonas aerugincsa . 1. 
Serotyping, Pyocine Typing and their Interrelations. Acta Pathol. 
Microbiol. Scand. 81: 70-80. 

Bergan, T. 1973. Epidemiology Markers for Pseudcmonas aeruginosa . 2. 
Relationships between Bacteriophage Siosceptibility and Serogroup and 
Pyocine Type. Acta Pathol. Micrcbiol. Scand. 81: 81-90. 

Bergan, T. 1973. Eipidemiological Markers for Pseudoroonas aeraginosa . 3. 
Comparison of Bacteriophage Typing, Serotyping, and Pyocine Typing on a 
heterogeneous Clinical Material. Acta Pathol. Microbiol. Scand. 81: 91- 
101- 

Bradbury, W.C. , A.D. Pearson, M.A. Marto, R.V. Congi and J.L. Penner. 1984. 
Investigation of a Campylobacter jejuni outbreak by serotyping and 
chromosamal restriction endonuclease analysis. J. Clin. Microbiol. 19: 
342-346. 

Bradbury, W.C, R.G.E. Murray, C. Mancini and V.L. Morris. 1985. Bacterial 
chramosomal restriction endonuclease ancilysis of the homology of 
Bacteroides species. J. Clin. Microbiol. 21: 24-28. 

Brown, M.R.V. and Foski, J.H. 1970. A simple diagnostic milk medium for P. 
aeruginosa . I of clinical pathology. 23: 172-177. 

Brutsaert, P. 1924. L'antigene des Baciles Pyocyaniques . C.R. Soc. Biol. 
Paris, 90: 1290-1292. 

Fisher, M.W. , Devlin, H.B. , and Qiabasik, F.J. 1969. New Immunotype Schema for 
Pseudomonas aeruginosa Based on Protective Antigens. J, Bact. 98: 835- 
836. 

Fluharty, D.M. and Packard, W.L. 1967. Differentiation of Gram Positive and 
Gram Negative Bacteria Without Staining. Amer. J. Vet. Clin. Pathol. 1: 
31-35. 

Galbraith, J.H. and Williams, R.E. 1972. Migration and Leaching of Metals from 
Old Mine Tailings Deposits. Groundwater 10: 33. 

Habs, I. 1967. Untersuchungen Uber Die 0-Antigene von Pseudomonas aeruginosa . 
Z. Hyg. 144: 218-228. 

Homma, J. Yuzuru, K.S. Kim, H. Yamada, M. Ito, H. Shionoya, and Y. Kawabe. 
1970. Serological Typing of Pseudomonas aeruginosa and its Cross 
Infection. Jap. J. Exp. Med. 40: 347-359. 

Kleinmaier, H. 1957. Die O-Gruppenbestinroung von Pseudomonas - Stammen Mittals 
objekttrager - Agglutination. Zbl. Bakl., 170: 570-583. 

Lanyi, B. 1966/67. Serological Properties of Pseudomonas aeruginosa . 1. Group 
Specific Somatic Antigens. Acta Microbiol. Acad. Sci. Hung. 13: 295-313. 



- 293 - 

Lanyi, B. Gregacs, M. , and Adams, M.M. 1966/67. Incidence of ?seudciTK:nas 
aeruqinosa Serogroups in Water and Human Feces. Acta Microbiol. Acad. 
Sci. Hung, 13: 319-326. 

Moraschi, T. , Bolles, D.M. , Moczulske, C, and Lindsay, M. 1966. Serological 
Types of Pseudononas aeruginosa Basfid on Heat Stable Antigens: 
Correlation of Habs' (European) and Verder and Evans' (North American) 
Classifications. J. Inf. Dis. 116: 84-88. 

Ringen, L.M. and Drake, C.H. 1952. A StLKly of the Incidence of Psei^omcnas 
aeruginosa frcan Various Natural Sources. J. Bact. 64: 841-845. 

Sandvik, 0. 1960. Serological Caiparison Between Strains of Pseudcincnas 
aeruginosa from Human and Animal Sources. Acta Pathol. Microbiol. Scard. 
48: 56-60. 

Seyfried, L-P. and Eraser, D.J. 1978. Pseudomonas aeruginosa in Swimming Pools 
Related to the Incidence of Otitis Externa Infection. H.L.S. 1591) : 50- 
57. 

Verder, E. and Evans, J. 1961. A Proposed Antigenic Schema for the 
Identification of Strains of Pseudanoncts aeruginosa . J. Inf. Dis. 109: 
133-193. 

Veron, M. 1961. Sur 1 ' agglutination de Pseudomonas aeruginosa : Subdivison des 
Groupes Antigeniques 0:2 et 0:5. Annales Inst. Pasteur, 101: 456-460. 

Wheater, D.W.F., Mara, D.D. , luzan Jawad and Oragui, J. Biological Lndicatcrs 
of Waste Quality A. James and L. Evison, (et) John Wiley and Sons (Pub) . 
1979. Ql. 21. 

Young, V.M. and Moody, M.R. 1974. Serotyping of Pseudomonas aeruginosa . J. 
Inf. Dis. J. 130 (S): S47-S51. 



- 294 - 
AHBOnX - G 

mi'lDOBfi^'IT^l? 1 1 JM SP. 

Bifidobacteria are gram-positive organisms which may have club-shaped, 
bifid ends. Colonies are about lnm in diameter, smooth, entire, convex, 
opaque, glistening and white. Bifidobacteria found in huinans include 3^ 
adolescentis . B. catenulatum . B. breve , B. lonqum . B. globosum and B. infant is . 
Even though they have been isolated, their significance has not been determined 
because biochemical fermentation reactions, and microscopic examination do not 
definitely differentiate between all Bifidobacterium species. Polyacrylamide 
gel electrophoresis of cellular proteins or other genetic investigations, which 
can give conclusive identification, can be time-consuming. 

The only pathogenic Bifidobacterium sp. is B. dentium which is found in 
the mouth, intestines, and in mixed infections of the Icwer respiratory tract. 
Because it is an obligate anaerobe, it produces acetic and lactic acids in 
peptone-yeast-extract-glucose broth and ferments many sugars. According to GIC 
results, bifidobacteria produce more acetic in comparison with lactic acid 
than other gram-positive, non-sporeforming anaerobic bacilli in humans. 

Bifidobacteria recovery from membrane filtration is hampered by the growth 
of other bacteria on the media. In YN17 (Mara and Oragui, 1983) , a media used 
for bifidobacteria cultivation, the antibiotics present prevent growth of other 
bacteria facilitating isolation and making identification easier. However, the 
addition of actidione (cycloheximide) to inhibit mold growth, and the addition 
of specific vitamins, minerails or other antibiotics, have been considered to 
fortify the media. 



- 295 - 

Bifidobacteria have t±ie ability to alter their appearance and this makes 
their classification difficult. 

Msfthcd of Recaverlna isoi^t-iac! 

Bifidobacteria isolates were obtained by diluting a sample and passing it 
through a membrane filter. 

On YN17 (blue) , bifidobacteria appears as very dark blue to black 
colonies. The green-tinged colonies are usually streptococci. Other media 
used included MRS (de Man et al . 1960) by Gibco or Oxoid. It was suggested 
that by the addition of cysteine hydrochloride, a reducing agent, the media 
becomes more anaerobic - thus helping to make the environment more conducive to 
this organism's growth requirements. YN17, without antibiotics or indicator, 
and TPY (recommended on Page 1423, Bergeys Manual, gram-positive organisms) 
were also used as growth media. Bifidobacteria responded to culture transfers 
and environmental stress by changing their cell morphology and biochemical 
reactions. 

Method of Analvsis 

The sample, either pure or from sewage, was diluted and tested by the MF 
procedure (i.e. sewage) or by spread plating (i.e. animal sources) . 

The plates were incubated anaerobically for 48 hours at 37° c using BBL 
anaerobic Gas Pack anaerobic systems (70304) and indicator strips (70504) in an 
anaerobic jar. 

Dark blue (YN17) colonies and circular, mucoid, white translucent colonies 
(TPY, MRS, 'mil) were ahosen for further examination. .\11 L-if creation 



- 296 - 

concerning growth, source, etc. was recorded. The colonies were labelled and 
inoculated into 5 ml MPS broth with cysteine hydrochloride added just before 
use. These tubes were incubated for 48 hours at 37° C anaerobically. From the 
tubes, isolated colonies were streaked on different media. The plates were 
incubated anaerobiccLLly for 2 days. Each isolate was streaked out on plates 
once more to ensure a pure culture. Ihe culture was tested with Gram reagent 
(3% potassium hydroxide) catalase reagent (3% hydrogen peroxide) and a slide 
was made of the organism using Crystal Violet. ( Bifidobacterium is gram- 
positive; the stain was used to observe cell appearance) . 

The following tests were applied for Bifidobacterium characterization: 
Gram stain, catalase, gelatin, bile esculin, Kligler's iron agar, sorbitol, 
xylose, cellctoiose, melezitose, arabinose, lactose, mannitol, melibiose, 
ribose, mannose, trehcLLose, raffinose, fructose and litmus milk (see Appendix) . 
All inoculated tests were incubated at 37*^ C in an anaerobic jar. The sugars 
were examined after 24 and 48 hours. A negative control for all the sugar 
reactions was incubated along with the other tests. The sugar tubes were 
discarded atfter 2 days. Other results were conclusive within 5-7 days. The 
appended flow chart was devised by Xu Yan, Arif Somani, R. Cesjardins to help 
in the analysis of the results. Please note that numbers on the flow chart 
correspond to Table 15.51 (Page 1428) Bergeys Manual, gram-positive organisms. 
Table 5 in the paper by Mitsuoka (1982) is also appended as a useful 
classification reference. 



- 297 - 







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- 298 - 



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- 299 - 
APPEldX - H 
FECKL SlEEETDGDOCr 

The following identification scheme (ASM, 1985) has been prepared by Gar/ 
Horsnell, Ministry of the Environment (1987) and has been employed at the 
Ministry of the Environment for fecal streptococcal identification - 

Introduction 

Ihe fecal streptococci of interest in this study may be illustrated by the 
tables given belcw (Modified by Hartman et al . 1966) . 

Pecal S Lx t ^jL ocoo ci 



Enterococci 



S. faecalis var faecalis 

S. faeccLLis var liquefaciens 

S. faecalis var zymogenes 

S . f aecium 

S. f aecium var casseliflavus 

S. durans 





S. bcvis 


Viridans 


S. equinus 


group 






S. mitis 




S. scdivarius 



Group D 



S. avium {GBP D, Q) 



MRdia 

The fecal streptococcus isolates were picked from recovery media such as m-E 
agar and m-Enterococcus agar. (Details of preparations can be found in the 
Appendix under Media.) Colonies of all sizes that appeared pink with a blue 
halo on m-E and pink to maroon on m-Enterococcus were chosen. Source, 



- 300 - 



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i i i M I 1 2. i 



^ n 



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I 



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>- + +• 
S 



- 301 - 

dilution, media were recorded as well. Care was taken to pidc (with a sterile 
loop or needle) only one colony for each isolate sainple. 

Method of Collecting Isolates 

The colony was streaked onto Brain Heart Infusion (Difco) agar slants and 
incubated for 18-24 hours at 37*^ C- The following day, the slants were covered 
with sterilized paraffin oil for storage. The isolate would no longer grew but 
would be kept free from contamination. 

Method of Identif icaticn 

Prior to inoculation, the EHI plates were dried under a laminar flow hood 
for approximately 15 minutes. The plates, labelled according to the isolate 
number, were streaked in 4 quadrants to obtain isolated colonies and incubated 
at Zl° C for 18-24 hours. The plates were then examined to determine how many 
different types of colonies were present by looking for a) colour; b) colonial 
morphology; and c) contamination. Each different type of colony, except the 
latter, was transferred onto a EKE plate divided into 6 sections to make a 
reservoir (i.e. a spread or patch of growth) . This was done to obtain a larger 
volume of bacterial growth for further testing. 

The next day, wet mounts of each sample were made and examined under the 
microscope. Streptococci are cocci, ovoid to spherical in shape. They occur 
in short chains (3-6 cells) on agar or longer chains (more than 6 cells) in 
broth or in pairs or individually. They do not appear as tetrads. Next, the 
following tests were performed to separate them from other gram-positive cocci. • 

Each isolate was tested with 3% H2O2 (hydrogen peroxide) for the catalase 
reaction. The test solution was first tested with known stock culture controls 
of S . f aecium and Pseudomonas aeruginosa to make sure the solution would 



- 302 - 

perforTn correctly. Both the catalase reagent and Gram reagent (KDH) were kept 
for 2 weeks. S . f aecium is catalase negative; i.e. no reaction when the 
culture is tested with reagent while P. aeruginosa is catalase positive. 
Introduction of culture to reagent produces biihhles. 

For the Gram test, potassium hydroxide (KDH) was used. The culture was 
mixed with a drop of NDH for about one minute with a loop. A gram-negative 
such as P. aeruginosa breaks down the cell wcLLl and the contents disperse. The 
mixture becomes thicker, syrupy and stringy when you raise the loop from the 
slide. A gram-positive culture (i.e. Streptococcus) will give no reaction with 
KDH. 

The isolate was tested on rabbit blood agar (see Appendix) with a single 
horizontal streak. The plates were incubated in an anaerobic jar using BBL 
anaerobic gas pack anaerobic systems 70304 and indicator strips 70504 for 43 
hours at 37° C (To ensure nedia efficiency, test with a known positive (i.e. S. 
faecal is v. zvmogenes ) and negative ( S. f aecium ) . A positive hemolysis will 
give a clearing of the media around the streaked zone. 

All results were recorded on bench sheets. Another isolate reservoir was 
strea k ed onto BHI. Much care was taken in the streaking process since blocd 
agar and EHI can be easily contaminated. 

Streptococci can be divided into four general groups: 

a) the viridans group 

b) the lactic group 

c) the pyogenic group 

d) the fecal or enteric group 

There are 6 tests used in this method to help to separate the fecal group 
from the others. These are: 

1. Bile esculin 

2. growth at 45° C - using Todd Hewitt broth (see Media section in 
Appendix) 

3. growth in 6.5% NaCl 

4. arginine 



- 303 - 

5 . haemolysis 

6. antigen serotyping (Group D) 

To identify an isolate as an enterococcal group D fecal streptococcus only 
the first four tests plus presence of the Group D antigen are required. 

The isolates were also inoculated into pyruvate, mannitol, sortose, lactose, 
melibiose, gelatin, litmus milk, arabinose and starch agar. 

The following observations and conclusions can be made fron the previous 
table: 

a) S . f aecium variants are usually cirabincse+-, pyruvate- and never gelatirn- or 
haeinolytic+. However S. f aecium var casseliflavus which is the only yellow 
pigmented organism in the enterococcal group can be arginine^-/-, pyruvate+/- 
, (and arabinose^-) . 

b) S . f aecalis variants are usually always pyruvate^-, arabinose- and may or may 
not be gelatirH", haemolytic+. 

It is pcssible for an organism to lose its ability to produce a positive 

result (i.e. to ferment a sugar) . Hcwever, an organism cannot gain an ability 

to react if it naturally did not have it before, unless the cell obtained a 

plasmid-carrying gene through interaction with other, similar organisms. 



- 304 - 
BinrfipnnfT^I ttp^i- Results 



Test 



Positive Reacticn 



Exanple/Notes 



bile 

arginine with paraffin oil 
(anaerobic) 



6.5% NaCl grcwth 
arabinose fermentation 
lactose fermentation 
pyruvate f ennentation 
mannitol fermentation 
sorbose 
litmus miUc 

gelatin 

Todd Hewitt 

melibiose 

blood 

starch agar 



blackening 




pink 


(all Group D are 

positive) except 

S. avium and sometimes 

S. faecium var 

casseliflavios 


grcwth 




yellow 


S. faecium 


yellcw 




yellcw 


S. faecalis variants 


yellow 


S. avium 


ciad, alkali 
curds/reduction 





liquefaction 



S. faecalis liquefaciens 
S. faecalis zymogenes 



grcwth 




yellcw 


S . faecium 


clearing 


S . faecal is 




zymogenes 



var 



S. bovis 



Serology 

By looking at the profile of all the results it was possible to make an 
identification for each isolate. If, by chance, the first 5 tests produced a 
negative result, it was necessciry to perform a serology test to see if Group D 
was present. 



- 305 - 

Group-specific antigens are usually carixihydrate structural ccitponents of 
the cell wall. Iliese antigens can be extracted in soluble form and identified 
by precipitation with hamologous antisera. 

There are a number of ways to extract these antigens from the cell wcLLl 
including: 

1. Hot HCl extraction 

2. Hot fonnamide extraction 

3. ■ Autoclave extraction 

4. Sonication, and 

5. Enzyme extraction 

Each extraction method has certain advantages and disadvantages. 
Generally the autoclave extraction method and the enzyme extraction method are 
simple yet reliable procedures for all groups including Group D. Some Group D 
streptococcus species (i.e. S. bovis . S. eouinus and S . avium ) , however, 
contain relatively snail amounts of this antigen and these may require more 
severe extraction procedures (i.e. sonication) . 

The antigen-antisera precipitation reactions can be performed in various 
ways including: 

1. Capillary precipitin test 

2. Slide agglutination reaction 

3. Electrophoretic methods. 

Probably the simplest method to employ is the slide agglutination 
procedure whereby group-specific antibody coated latex particles are reacted 
with the antigen extract. 

There are commercially prepared kits available which provide the enzyme 
for an enzyme extraction, a reaction slide and antibody coated latex particles 
for various serogroups (generally groups A, B, C, D, F, & G) . These latex 
particles can ailso be reacted with extract frcrn any other extraction procedure. 



- 306 - 
The methcxi is as follows: 

1. A pure isolate is inoculated into EHI broth and incubated for 18-24 hours 
at 37° C. ("Hie broth is brought up to 1% glucose by adding 3g/l and put 
in suitable screw cap tubes for centrifugation. ) 

2. Next day, the broth, culture is centrifuge! at 3000 rpra for 10 minutes to 
pack the cells. 

3. Hie clear liquid broth (supernatant) may be reacted with the group D 
antisera (Group D antibody coated latex particle suspension) (Streptex 
Latex Group D suspension Wellcome Diagnostics) to determine the presence 
of the Group D antigen. A drop of the suspension is placed on a tile to 
which a drop of supernatant is added. "Hie tile is placed on a Variable 
Speed Rotator. Observation is made. If agglutination occurs, the 
reaction is recorded as a Group D positive. If it doesn't take place, the 
extraction procedure continues. 

4. The supernatant is carefully decanted from the tube and put in 
disinfectant. 

5. Five 5inL of physiological sciline (0.35% NaCl) is added to the tube to 
resuspend the cells. 

6. The tube is once again centrifuged at 3000 rpm for 10 minutes to pack the 
cells. 

7. The clear saline is decanted into a disinfectant (Dettol) . 

8. Then 0.4 ml extraction enzyme (Streptex Extraction Enzyme S . griseus 
Wellcome Diag. ) is added to the tube which is shaken to resuspend the 
cells. 

9. The tubes are incubated at 37° C (water bath) for 1 hour. 

10. Following this, the tubes are autoclaved for 30 minutes 15 psi (121° C) . 

11. The tubes were cooled and centrifuged at 3000 rpm for 10 minutes. 

12. The clear liquid phase is tested for the presence of the group D antigen. 
1 drop clear extraction liquid is added to 1 drop group D latex 
suspension. A group D positive reaction is indicated by agglutination 
(granulcir appearance in mixture) . If the mixture remains clear, the 
isolate is group D negative. 



- 307 - 

AtVtMlLX - I 
PSEDEOCHaS AEPDGINCSA . 

Introducticri 

Pseudcmonas aeruginosa can be found in soil, water, sewage, plants and the 
mammalian gut. The organism is pathogenic for hxjmans, plants, insects and some 
animals. Since nutritioncLL requirements are simple, and the organism can majce 
use of organic ccnpounds, and exist in water of ambient temperature, 
Pseudomonas aeruginosa is commonly found in hospitcLL wards. Pseudomonas 
aeruginosa produces many toxins and enzymes. 

Since Pseudomonas aeruginosa is an opportunistic pathogen, and has been 
the cause of severail infections carried through contaminated waters to a 
vulnerable host. Pseudomonas aeruginosa has been shown to be the cause of 
outer ear infections in swimmers (Seyfried and Fraser, 1978) . 

A count of 10 organisms/ 100 ml indicates recent fecal pollution (I^E, 
1983) . 

The medium used for Pseudomonas aeruginosa enumeration is m-PA (Levin and 
Cabelli) (Appendix) . The antibiotics ;:sed in the medium and the high 
incubation temperature, help make the medium selective for Pseudomonas by 
inhibiting growth of other hete rot r o phic bacteria. 

Method of Sanpling 

The sample was tested using the membrane- filtration technique and 
dilutions were made as required. 

The m-PA plates were incubated for 48 hours at 41.5 + 0.5° C. The plates 
were placed, inverted, in a cakette moistened with paper toweling. 



- 308 - 
Pseudomonas colonies appeared flat, spreading, brownish-green, or tan. 
Typical colonies were confirmed by streaking them on Skim Milk Agar (Appendix) 
with incubation at 37° C for 48 hours. A positive result was indicated by a 
clearing of the n^yjiq and a fluorescent pigment. "Hie culture was then 
transferred onto a BHl slant and numbered. 

Analysis of PsaJdancnas Isolates 

The isolates were streaked fran the BHl slants onto nutrient agar (Difco) 
to obtain isolated colonies. These plates were incubated 18-24 hours at 37° C. 
The next day, the plates were examined to determine whether the plate had only 
one type of colony. If the organisms on the plate appeared as a pure culture, 
the colonies were tested with: 

a) Gram reagent which was tested against a known positive and negative 
control. For example. Streptococcus ffaecium ) (G+) was used as a negative 
(not sticky) and our Pseudomonas aeruginosa (G-) control was used as a 
positive (sticky, syrupy) . 

b) Oxidase reagent. On a filter paper (Whatman #1) which was cut to fit 
inside a petri dish, the oxidase reagent was applied to the paper and 
allowed to dry. (If the oxidase appeared blue from the storage bottle, it 
was discarded) . A colony was taken from the plate with a wooden 
applicator and stamped onto the paper. A positive reaction resulted when 
the spot turned blue, violet. This occurred a few minutes after being 
tested. Results were recorded. In addition, the culture was also 
streaJced on acetamide slants and skim milk agar. 

The acetamide tubes were incubated at 37° C for 72 hours. A positive 

appeared as bright pink. 

The skim milk agar comprised 4 different tests: 

a) A cleeiring of the medium surrounding the streaked zones (+) ; 

b) The culture appeared to fluoresce under an Ultra Violet light source 
(short wavelength) . These stains produced water-soluble pigments 
(pyoverdins) . Pyoverdin production is dependent on nutritional factors. 



- 309 - 

c) The growth on the plate appeared as yellow-green to brown (pyocyanin 
pigment production) +. 

The pyocyanin production, which is a blue water-soluble non- fluorescent 
pigment is excreted by Pseudoncnas aeruginosa into the medium. 

The greenish appearance is caused fay the presence of the (blue) pyocyanin 
and the yellcw pyoverdins. 

d) Pseudomonas aeruginosa strains possess a grape-like odour. 



Sanple Prepeiraticn for Serotyping 

1. All cultures showing positive restiLts were sub-cultured to reservoirs on 
Nutrient agcir (Difco) with a sterilized swab. 

Incubation of the inverted plates was for 18-24 hours at 37<^ C. 

2. From the reservoirs, thick suspensions of growth were made into 10 mL of 
sterile 0.85% NaCl (Appendix) . The tubes were autoclaved for 30 minutes 
at 121° C with a slow exhaust. 

3. The autoclaved suspension was centrifuged at 1000-2000 rpn for 10 minutes 
and the supernatant was discarded. 

4. The pellet was resiispended in 0.75 mL of a solution consisting of 0.85% 
NaCl and 1/10,000 merthiolate (Appendix). 

Dilution of Sera 

The following steps were followed using the 17 sera from the Pseudomonas 
aeruginosa antisera kit (Difco) : 

1. Resuspended sera by adding 1 mL sterile distilled water to dissolve 
contents. 

2. Sterilize approximately 20 screw cap tubes. 

3. Pipet 0.9 mL of the 0.35% NaCl -merthiolate solution into each tube. 
(label tubes 1-17) . 



- 310 - 

4. Using a clean pipet each time, pipet 0.1 mL of sera into the appropriate 
tube. 



Testing the Sera 

Every day, before the samples were tested the following procedure was followed. 
(Both positive and negative controls were tested first) . 

1. Antiserums 1-17, as already described, were diluted 1:10 (0.1 mL antiser:a 
-I- 0.9 mL NaCl and merthiolate solution) . 

2. Antigens 1-17, which were already in solution were mixed (0.1 mL antigen 
and 0.9 mL rabbit sera*. *Ratbit serum was first diluted 1/10) . 

Positive Oontral 

1. Place a drop of antiserum 1 on tile. 

2. Place a drop of antigen 1 on top. 

3. Next, put the tile on a Variable Speed Rotator (Yankee 070504, Clay Adams, 
Parsippany, MJ) so that it will rotate gently. Leave for a few minutes. 
There should be agglutination. If however, there isn't, use the antigen 
full strength (not diluted with rabbit serum) . Lack of agglutination can 
occur if there is too much organism present. A prozone is created 
(meaning an excess of antigen) , thus no agglutination occurs. 

4. Each antisera should be tested with its corresponding antigen. 



Testing Collected Isolates 

1. Place a drop of each antisera onto a separate square of the tile. 

2. Add the diluted, prepared and mixed sample - one drop per square. 

3. Record tile and sample number for referral. 

4. Place on the Rotator. 

5. Observe closely after a few minutes for agglutination (forming of 
granules) . 

6. Record results. 



- 311 - 
Negative CJcntrol 

1. A drcp of Antiserum 1 is mixed with a drop of antigen 2. 

Result 
Antigen 1 + Antiserum 1 + 

Antigen 2 + Antiserum 1 



Antigen 2 + Antiserum 2 
Antigen 1 + Antiserum 2 



Antigen 3 + Antiserum 3 + 

Antigen 1 + Antiserum 3 
(or any except 3) 

It is important that no agglutination occurs among heterologous mixtures. 

Antisera can be kept in t±ie refrigerator (when not in use) for 5 days. 

Antigens made with rabbit sera should be prepared fresh daily. 

Iferthiolate (Thimerosol) is a preservative and antiseptic used to prevent 
contamination of the antisera. 

All antigens and antisera are tested in this manner. 

Precauticns 

1. The diluted sera should not be kept longer than 5 days. 

2. The 0.35 NaCl + merthiolate solution shouldn't be kept longer than 5-7 
days unless it is frozen. 

3. Unused NaCl and merthiolate solution may be frozen. All unused reagents 
should be refrigerated. 

4. Note which sera gives weak reaction with antigen control; i.e. 2,3 give 
poor reactions when testing with these sera. 

. Make sure you observe the sera mixture quite closely from the time you 
place the drops on the tile for at least one minute. 

5. Keep tile gently swirling at all times. 



- 312 - 
APPENDIX - J 

MEDIA EraPARATICN 
Acetamide 

Arabinose (see CS method) 
Arginine 
Bile esculin 
(Rabbit) blood agar 
1% Carbohydrate solutions (CS) 
Catalase reagent 
Crystal Violet Reagent 
Gelatin 
Grains Iodine 
Gram Reagent 

Lactose (see CS. method) 
Litmus milk 
Magnesium chloride 
Mannitol (see CS. method) 
mr-CP2 
m-E 

Melibiose (see CS. method) 
m-Enterococcus 
m-PA 
MPS agar 
m-Tec 

0.85 NaCl and merthiolata solution 
Oxidase reagent 
Phosphate buffer, stock solution 



- 313 - 

Riosphate buffer, dilution blanks 

Pyruvate broth 

Saline 

Skim milk agar 

6.5% Sodium chloride 

SortxDse (see C.S. method) 

Starch agar plates 

Todd Hewitt broth 

TPY 

Urease reagent 

YN17 

Medium - Acetamide Agar Slants 

Ingredients: 

NaCl 5 . Og 

Dipotassium hydrogen phosphate (K2HPO4) 1.4g 

Acetamide 10 . Og 

KH2PO4 . 7g 

Mg SO4.7H2O l.Og 

Hrenol Red 0.012g 

Difco Bacto-Agar* 15. Og 

Distilled water lOOOmL 

Preparation: 

- * Add all ingredients except agar 

Adjust pH to 6.8; then add agar 

- Heat to 92° C 

Autoclave media, empty screw cap tubes, screw caps and syringe at 121° C 
for 15 minutes 

- Before dispensing under laminar flow, adjust pH to 6.8 i_ 0.2 
Slant tubes to dry 

Reading: 

- The acetamide test is used for Pseudcmcnas aerugincsa identification. 
The slant is streaked along the surface and incubated for 3 days at. 35° 
C. A bright pink is indicative of a positive reaction 



- 314 - 

Medium - Ar^ginine Dihydrolase Medium Clhomley) 

Ingredients: 

Bacto Peptone (Difco) 1-Og 

NaCl 5-Og 

K2HPO4 0-^g 

LrArginine Hydrochloride 10. Og 

Ehenol Red 0.01 g 

Bacto Agar (Difco) 3.0g 

Distilled Water lOOOmL 

Preparation: 

- Mix ingredients into Distilled water 

- Adjust pH to 6.8 if necessary 

- Heat to 90°C, to dissolve 

- Dispense into tubes (5ml/tube) 

- Autoclave 15 min. , 15 psi (121° C) 

Inoculation: 

- Using a needle pick up growth from plate and stab straight down through 
centre of arginine medium. Layer top of Arginine medium with 
approximately 1 cm sterile paraffin oil. 

Incubation: 

- 35° C, up to 5 days 
Reading: 

- positive, medium changes from orangey-pink to bright pink at any tinie up 
to 5 days 

- negative, no change 

Medium - Bile Esculin Agar 

Ingredients: 

(Difco) Peptone 5.0g 

(Difco) Beef extract 3.0g 

(Difco) Oxgall 40. Og 

(Sigma) Esculin l.Og 

Ferric citrate 0.5g 

(Difco) Agar 15. Og 

Distilled water 1000 mL 



- 315 - 

Preparation: 

- Mix ingredients into Distilled water 

- Heat to 90° C, to dissolve 

- Dispense into tubes with screw caps (5 ml/tube) 

- Autoclave 15 min. , 15 psi (121° C) 

- Cool tubes in slanted position 

Inoculation: 

- Transfer growth from plate and smear, with loop, over slant 

Incubation: 

- 35° C, up to 72 hr. (3 days) 

Reading: 

- Positive, blac3csning of one-half or more of slant at any time up to 72 
hr. 

- Negative, blackening of less than one-half or no blackening of medium at 
72 hr. 

Medium - Blood Agar (Rabbit) (5%) 

Ingredients: 

(Difco) Blood Agar base (powder) (BAB) 40. Og 

Rabbit Blood (Defibrinatad) 50 mL 

Distilled Water 1000 mL 

Preparation: 

- Mix BAB powder into Distilled Water 

- Heat to 90° C, to dissolve 

- Autoclave 15 min. 15 psi (121° C) 

- Cool to approx. 50-55° C 

- Aseptically add 50 mL defibrinatad rabbit blood 

- Pour plates under laminar flow hood 

Inoculation: 

- 1. Pour plate + aerobic incubation 

2. Streak plate + anaerobic incubation 

Incubation: 

- Suggest, 35° C streak, anaerobic, 48 hours 



- 316 - 

Reading: 

- Three Haeinolytic reactions possible 

1. Beta (B) - Complete clearing around colony 

2. Alpha (a) - Incomplete clearing around colony with red cells left 

intact close to colony 

3. Gamma (t) - No Haemolysis 

Rai±iit blood is used for hemolysis testing because, even though Streptococci 
can produce a positive result on other animal bloods, rabbit blood helps to 
differentiate between the fecal streptococci since only S. Faecalis z'/r^ccer.es 
produces B haemolysis. 

Medium - 1% Cartxshydrate Solution 

Ingredients: 

(Difco) Heart Infusion Broth (powder) 25. Og 

Cartx±iydrate Sol^ (10% Aqueous) 100 mL 

Distilled Water 900 mL 

Brom Cresol Purple Solution (BCP) 1.0 mL 

Preparation: 

1. Stock indicator Solution 

1.6g BCP in 100 mL 95% EIOH 

2. Carbohydrate Solution 

Carbohydrate 10. Og 

Distilled Water 100 mL 

- Mix HIB powder into 900 mL Distilled Water 

- Add Carbohydrate Sol^ (100 mL) 

- Add BCP indicator (1 mL) 

- Dispense into tubes (5 mL/tube) 

- Autoclave 10 min, 15 psi (121° C) 

Inoculation: 

- Using a needle inoculate broth tube 

Incubation: 

- 35° C, up 72 hr. (3 days) 

Reading: 

- Positive, colour change from purple to yellow 

- Negative, no change 

The above recipe can be followed to make the following sugar solutions: 
lactose , mannitol . 



- 317 - 

Sugars such as arabinose, sorbose, melibiose are made slightly differently. 

Make the HIB solution with indicator. 

Autoclave it with test tubes and caps (separately) and syringe. Filter 
sterilize the carbohydrate solution (2) into the HIB after it has cooled. 
Syringe the solution into the tubes under the laminar flow. 

The media is tested with a positive and negative to check it is working 
properly. 

The tubes are inoculated with some culture and checked every day for 5 days for 
positive results and recorded. After incubation time, the result is recorded 
as negative if media remains purple. 

Medium - Catalase Solution 3% Reagent 

Ingredients: 

H2O2 (hydrogen peroxide) 30% ' 5 mL 

Distilled Water 45 mL 

Preparation: 

Keep refrigerated for maximum 2 weeks. Check before use, to make sure it 
works properly. 

Reading: 

- [Jrops of this reagent are placed on a microscopic slide. The solution is 
firstly tested with known cultures which would produce positive (bubbles) and 
negative (no bubbles) results when the culture is added (no mixing necessary) 
to the reagent. 

Medium - Crystal Violet Reagent 

Ingredients: 

A Crystal violet 2.0g 

Ethanol, 95% 20 mL 

B Ammonium Oxalate 0.8g 

in 80 mL distilled water 

Preparation: 

- Add A to B 

- Filter 



- 313 - 

Medium - Gelatin (12%) • 

Ingredients: 

(Difco) Heaort Infusion Broth (powder) 25. Og 

(Difco) Gelatin 120. Og 

Distilled Water 1000 mL 

Preparation: 

Mix ingredients into distilled water 
NB - Add Gelatin very slowly so as not to cause clunping 
Heat to dissolve (90° C) 
- Dispense into tubes (5 mL/tube) 
Autoclave 15 min, 15 psi (121° C) 

Inoculation: 

- Using a needle stab inoculate through Medium 
Incubation: 

- 35° C, for 5 days 
Reading: 

- After incubation remove all tubes to refrigerator (-10° C) , cool until 
uninoculated control tube is solid when inverted. (15 min. in 
refrigerator) 

- Positive, medium remains liquid when inverted after cooling 

- Negative, medium remciins solid when inverted after cooling 
Medium - Gram's Iodine 

Ingredients: 

Iodine Crystals l-Og 

Potassium Iodide 2.0g 

Distilled Water 300 mL 

Preparation: 

- Dissolve chemicals in a small amount of distilled water. Make up to 300 
mL. 

Medium - Gram Reagent, 3% KDH 

Ingredients: 

Potassium Hydroxide l-5g 

Distilled Water 50 mL 



- 319 - 

Application: 

- TJiis reagent should be kept maximum 2 weeks refrigerated and al'.v-ays 
tested prior to use. 

- The reagent should first be tested with (2) controls which would give a 
pcsitive and negative result to make sure it is working properly. 

- Drops of the reagent are placed on a microscope slide. 

- A loop of culture is mivpri with the reagent for approximately 60 seconds. 

Reading: 

- A Gram positive result (G+) occurs when your mixture does not thicken or 
become stringy when you lift the loop from the mixture. 

- A Gram negative culture when mixed with WDH becomes syrupy or mucoid. 
When the loop is raised frcm the mixture it adheres and becomes stringy. 

Medium - Litmus Milk 

Ingredients: 

(Difco) Litmus Milk Powder 100. Og 

Distilled Water 1000 mL 

Preparation: 

- Weigh litnus milk powder and add it to the distilled water. P-it it on 
hot plate just to dissolve. 

- Dispense 5 mL per test tube. 

- Autoclave for 15 min, 121'C. 

The litinus milk test was performed on all fecal streptococci isolates. The 
culture was inoculated into the tube and incubated at 37° C and checked for up 
to 7 days. 

There are many reactions which can occur in litmus milk (which is a mauve, 
when uninoculatad) 

Reduction - The solution or a part of such will turn straw-like (beige) 
Acid Curd - Reduction usually occurs in acid curds. Plus there is a 

solid curd, pellet on the bottom of the tube whiah does not 

move. There, also, may be a clear whey-like liquid en the 

top. 
Alkaline Curd - Reduction usually occurs in adkali curds as well. There is 

a much softer, pliable curd on the bottom '..*u.ch can move or 

run. And on the top, there is a red to maroon liquid, 

opaque phase. 
Proteinization - Sometimes, what was once an alkali curd can become all 

liquid (may be same colour and texture as the top phase m 

an alkali curd) . 

Medium - Stock Magnesium Chloride Solution 



- 320 - 

Ingredients: 

^SgCl2 38. Og 

Distilled Water 1000 mL 

Preparation: 

- Dissolve MgCl2 in the distilled water. 

- Keep refrigerated. 

This solution is used to make buffered dilution blanks (along with using stock 
Phosphate Buffer solution) . 

Use 1.25 mL stock Phosphate Buffer and 5 mL Stock iMagnesium chloride solution 
per litre of distilled water. 

Dispense into bottles and autoclave 15 min, 15 lb. 

Medium - m-CP2 

Ingredients: 

(Difco) SFP Agar Base 52. 5g 

L-cysteine Hydrochloride 0.5g 

Distilled Water 1000 mL 

Preparation: 

- Dissolve at boiling temperature 

- Autoclave at 121° C for 15 min. 

- Cool to 50° C and then filter sterilize (separately) : 

sodium resazurin 2mg 

Neomycin . 15g 

Sodium Azide 0.2g 

indoxyl-B-D-glucoside . 5g 

Reading: 

- Target colonies (i.e. Clostridium perfringens ) are yellow with a black 
centre which can extend to circumference and are not surrounded by a blue 
halo. 

Medium - (IXifour's Modified) m-E 

Ingredients: 

Peptone ICg 

Yeast Extract 30g 

Sodium Chloride 15g 

Sodium Azide 0.15g 

Actidione (= cycloheximide store at 4° C) O.OSg 

Agar, Bacto (Difco) 15g 

Distilled Water 1000 mL 



- 321 - 



Preparation: 

Heat to 90° C. 

Autoclave for 15 min. , 121° C. 

After avitoclaving, cool the media to 55-60° C 

Add ascepticallv . Add all separately: 

for 1000 mL 

Nalidixic Acid 240 mg in 3 ml sterile distilled '^rater and 0.2 

ml ION NaOH, mix and add to medium 

Indoxyl-B-D-glucoside 500 mg in 5 ml ethanol (95%). Mix well; then 

add 5 ml distilled water and add to medium 

Triphenyltetrazoliura 

chloride (TIC) 20 mg add jxjst before pouring. 

Add very last ' 

Final pH 7.1 + 0.1 (adjust to 7.1, if necessary) 

Pour media under a laminar flow into square petri di s h es 

This media is used to enumerate and isolate fecal streptococci. 

Membrane filtration is performed on a sample. The filters are placed on the 
media (which, because of its very high salt concentration makes it very harsh 
on the colonies) . The plates are incubated inverted at 41.5'° C for 48 hr. in 
a cakette, moistened with paper towelling. 

Target colonies appesir as a pink button surrounded by a blue hcilo. 

Medium - m-Enterococcus Agar 

Ingredients: 

(Difco) Bacto m-Enterococcus Agcir 42. Og 

Distilled Water 1000 mL 

Preparation: 

- *Sterilize spatulas and distilled water with magnetic stirrer for 15 min. 
at 121° C 

- Allow to cool 

- Weigh out m-Enterococcus agar. Flame beaker before adding media to water 

- Sterilize a thermometer by immersing it in ethanol and wiping it dry. 
Place inside media 

- Heat media to 90° C 

- Once media reaches 90° C, change ever to another (cold) hot plate. Quick 
cool the media by placing it in a cakette with ice. Keep changing the 
ice to bring the temperature down rapidly. 



- 322 - 

- When tlie media has reached 55-60° C, check the pH, adjust (if necessarv') 
to 7.2 + 0.2. Pour the media under a laminar flow hood into square petri 
dishes. 

This media is used to enumerate and isolate fecal streptococci. The media is 
placed inverted in a cakette which has been moistened with paper towelling. 
Plates were incuisated at 37'° C for 48 hours. Target colonies may be pink to 
maroon, and of any size. The plates with approximately 8-150 colonies were 
counted. With the presence of 2,3,5 - Triphenyl-Tetrazolium chloride (TTC) all 
streptococci appear as pink, maroon, along with background or undesired 
colonies. 

without using TTC strep, faecium and strep, faecal is variants all appear pink, 
maroon. However strep, faecium casseliflavus appears yellow. 

Medium - MOE Formula mPA Agar (Sterile Technique) 

Use sterile glassware 



L-Lysine Monohydrochloride 

Yeast Extract 

Xylose 

Sodium Thiosulphate 

Magnesium Sulpiiate, Anhydrous 

Sucrose 

lactose 

Sodium QiLoride 

Ferric Ammonium Citrate (green) 

Sodium Desoxycholate 

Kienol Red 

Distilled Water, Sterilized 

(Difco) *Agar Bacto 

*Adjust pH to 7.60. Then add agar. 

Preparation: 

- Heat to 93° C until boiling starts to dissolve agar. 

- DO NOT AUTOCLAVE 

- Cool to 60° C. Add antibiotics and mix thoroughly 

- Antibiotics are dissolved in 200 mL sterile distilled water 

Antibiotics Amount per litre 



5.0g 
2.0g 
. 2.5g 
5.0g 
1.5g 
1.25g 
1.25g 
5.0g 
0.80g 
O.lOg 
O.OBg 
800 mL (to make 1 litre) 
15. Og 



Sul f apyr idine 
Kanaitiycin sulphate 
Nalidixic acid 
Actidione (Cycloheximide) 



0.176g 
0.0085g 
0.037g 
O.lSOg 



- Heat antibiotic mixture to approximately 50° C (no higher) to help 
dissolve (if necessary) . 

- Check surface pH, adjust, if necessary, to 7.1 

- Cool to 50° C (no higher than 60° C) 

- Pour into petri dishes 

- Final pH 7.1 + 0.1 



- 323 - 

This media is used for enumerating Pseudomonas aerjainosa count. 
Moistened paper towelling is added to a cakette, inverted plates are placed 
inside and incubated at 41.5° C for 48 hr. Target colonies may appear brovvn, 
black. Tan colonies from the filter are streciked on Skim Milk Agar for 
Ps. aeruginosa confirmation. 

Medium - MRS Agar Plates 

Ingredients: 

*Cysteine Hydrochloride 0.3g 

Gibco MRS Etoth 52. Og 

Difco Agar, Bacto 15. Og 

Distilled Water 1000 mL 

E>reparation: 

- Dissolve 52. Og MRS broth into 1000 mL distilled water. 

- Add 15 gm agar. Heat to 90° C to dissolve. 

- Autoclave for 15 min. at 121° C. 

- After autoclaving, cool media to 55-60° C. 

*Asceptically, add cysteine hydrochloride. 

- Pour media under a laminar flow hood into round petri dishes 100 mm x 
ISram. 

This media was used to isolate, cultivate Bifidobacteria eit.her by spread 

plating or membrane filtration. 

Plates were incubated anaembically in an anaerobic jar using BEL GAS Pack 

anaerobic systems (70304) with BBL Gas Pack indicator strips (70504) for 24- 

48 hr. at 37° C. 

Colonies appeared white, round; opaque and mucoid. 

Medium - m-TEC Agar (Thermotolerant E^ coli ) 

Ingredients: 

Proteose Peptone No. 3 

Yeast Extract 

Lactose 

Sodium Chloride 

Potassium Phosphate, Dibasic K2HPO4 

Potassium Phosphate, Monobasic, 

Sodium Lauryl Sulphate 

Sodium Deoxycholate 

Brcmocresol Purple 

Bromo Phenol Red 

Difco Agar, Bacto 

Distilled Water 





5.0g 


Difco 




3.0g 


Difco 




10. Og 


BCH 




7.5g 


BEH 


[PU4 


3.3g 


Fisher 


KH2P04 


l.Og 


Fi sher 




0.2g 


BCH 




O.lg 


BOi 




0.08g 


Difco 




0.08g 


BEH 




15. Og 


Difco 



- 324 - 
Preparation: 

- Mix all ingredients into 1000 mL distilled water. 

- Heat to 90° C to dissolve the agar. 

- AL±oclave 15 min./lS P.S.I./121° C. 

- Allow to cool to 50° C after autoclaving and pour, asceptically into 
sterile plates. 

- Firal surface pH should equal 7.1 + 0.1. 

Because nt-Tec media is the least selective of the ledia used, a negative 

control (passing sterile phosphate buffer through unit) was placed on media 

before beginning each new saitple. This ensured the unit and buffer were 
sterile. 

This media is used for enumerating fecal coliforms and E. coli . 

Moistened paper towelling is ridded to a cakette, inverted plates are balanced 
in the centre and a jar, frozen with 50 mL tap water is placed at either end of 
catette. Cakette is incubated at 44.5° C for 23 ± 1 hr. 

Yellcw-yellow green colonies aire counted from a filter with 10-150 colonies 
present. 

Medium - 0.35% NaCl and Merthiolate Solution 

Ingredients: 

Merthiolate (refrigerated) O.Olgm 

NaCl .35gm 

Distilled Water 100 mL 

Preparation: 

- Dissolve merthiolate, NaCl in distilled water. 

- Autoclave for 15 min. , 121° C. 

- Refrigerate, when cooled. 

This solution is used as a diluting reagent for Pseudomonas Antibody Sera. 

To dilute sera (i.e. antisera) 0.9 mL of the above solution is mixed with 0.1 
mL sera (i.e. antisera) . 

To prepare Pseudomonas aeruginosa isolates for serotyping, after a pure culture 
is grown in saline overnight, it is autoclaved and centrifuged. The above 
solution (0.75 mL) is added to the pellet and mixed. And this solution is used 
for tge slide agglutination test. 

M edium - Oxidase Reagent 

NNN%^ - Tetramethyl - p - Phenylenediamine 0.25g 
Ascorbic Acid 0.025g 

Sterile Distilled Water 

(warm) not higher than 35° C 25 mL 



- 325 - 

Preparation: 

- NEVER STIR - GENTLZ SHAKE (I.E. RDIAIE) 

- * Add ascorbic acid to distilled water to give a 0.1% solution. 

- Add 0.25g of NNN%-^ - Tetramsthyl - p - Phenylediamine dihydrochloride 

- Store in dark, 30 mL eye dropper bottles at 4^ C. 

- Reagent should last 7-14 days, but should not be used if it turns blue. 

Hie oxidase reagent drops cure applied to a Whatman No. 1 filter paper --vhich has 
been cut to fit a petri dish. The oxidase test is performed once the paper has 
dried. The reagent is firstly tested with cultures which would produce a 
positive result, and a negative result. A wooden applicator is used to pick 'jp 
some culture and pressed on the oxidase paper. A positive is indicated if the 
spot turns blue (purple) ; while a negative appears as no change. 

Medium - Phosphate Buffer Stock Solution 

Ingredients: 

Potassium dihydrogen phosphate (KH2PO4) 34. Og 
Distilled Water volume up to 1 L. 

Preparation: 

1. Dissolve KH2PO4 in 500 mL distilled water (in 2 L. beaker) 

2. Adjust pH to 7.2 + 0.2 with l.Oti NaOH. 

3. Pour this amount into 1 L. graduated cylinder and make up to 1 L. volume. 

4. Pour the solution back in to the same beaker and autoclave it at 121° C 
for 15 min. 

After autoclaving, when cooled, pour the sterilized solution in to a sterilized 
1 L. volumetric flask. 

May be kept at 4° C for no longer than 1 month. 

The above stock solution is used to make: 

a) Wash waters for membrane filtration (MF) 

Use 1.25 mL stock solution per 1 L- distilled water 

Autoclave 15 min., 121° C. 

Refrigerate. 

b) Dilution blanJ<s for samples for MF or spread plating. Use 1.25 mL stock 
phosphate buffer solution and 5 mL stock magnesium chloride per L. 
distilled water. Fill dilution blanks with approximately 104 mL/bottles 
(99 mL + 4 mL for evaporation) . Autoclave for 15 min. at 121° C. 
Refrigerate. 



- 326 - 

Medium - Pyruvate Broth 

Ingredients: 

Tryptone 10. Og 

Yeast Extract 5.0g 

K2HPO4 5.0g 

NaCl 5.0g 

Pyruvic acid, sodium salt 10. Og 

Bnanothymol blue (BTB) 0.04g 

Distilled Water 1000 mL 

Preparation: 

- Mix all ingredients except BTB into distilled water. 

- * BTB solution 

Mix 0.5g bramothymol blue in 5 mL IN NaOH . 
Add 0.4 mL of this solution into basal medium. 

- Adjust pH to 7.2 

- Dispense 5 mL per tube 

- Autoclave 15 min. at 121° C 

The streptococcus culture is inoculated into pyruvate broth. The test tube is 
inoculated for 24 hours (only) at 35° C. A lime green or yellow represents a 
positive result. 

Medium - Saline (physiological, 0.85%) 

Ingredients: 

Sodium chloride 8.5g 

Distilled Water 1000 mL 

Preparation: 

- Dissolve scilt in the distilled water. 

- If required in test tubes, dispense before autoclaving. 

- Autoclave at 121° C, 15 lb. 

- Ke ep refrigerated. 

Medium - Skim MiUc Agar 

Ingredients: 

Difco Slcim Milk Powder lOOg per litre 

Difco Bacto-Agar 15. Og 

Distilled Water 1000 mL 



- 327 - 
Preparation: 

- Slowly add skim milk powder to 500 mL distilled water and stir without 
heat for 30 min. 

- In a separate beaker, slowly add the agar to 500 mL distilled water vvtiile 
stirring. Heat the agar solution slowly to 90-92° C (10-12 min.)* 

- Autoclave the two solutions separately for 12 min. at 121° C. After 
autoclaving, cool the 2 solutions to approximately 55' C then [at which 
time, add the skim milk solution asceptically to the agar solution. Stir 
the mi>:ed solution for an additional 2-3 min. (Temperature should be 50- 
52° C) ] . 

- Dispense asceptically into sterile plates 
[20 mL in round (100 x 13 inm) plates]. 

- Prepare a small plate for quality control pH reading. 

- Adjust to 6.4 + 0.2 at 25° C (surface reading) . 

This media is used to determine many characteristics associated with 
Pseudomonas aeruginosa . such as pigmentation, caseinase production, 
fluorescence and oxidase reaction. Plates are streaked for isolated colonies 
and incubated for 48 hr. at 37° C. 

Pseudoinonas aeruginosa usually appears yellow-green, lime green, dark green, 
brown on Skim milk agar. 

A clearing of the media indicates caseinase production. Under ultra-violer 
light source, Pseudomonas aeruginosa colonies fluorese. Using the plate, 
provided the sarnple appears pure, it can be tested with the oxidase reagent 
(Ps. aeruginosa +) (Please see oxidase Reagent for detailed information) . 

Medium - 6.5% Sodium chloride 

Ingredients: 

(Difco) Heart Infusion Broth (powder) 25. Og 

NaCL 65 . Og 

Distilled Water 1000 mL 

Preparation: 

- Mix ingredients into Distilled Water 

- Dispense into tubes (5mL/tube) 

- Autoclave 15 mi, P.S.I. (121° C) 

Inoculation: 

- Using a needle pick some growth frcm the plate and inoculate lightly into 
the broth - no visible turbidity should be detected after inoculation. 

Incubation: 

- 35° C, up to 72 hr. (3 days) 



- 323 - 
Reading: 

- Positive, growth (visible turbidity) at any time up to 3 days 

- Negative, no visible turbidity at 3 days 
Madium - Stcirch Agar Plates 

Ingredients: . , ., 

(Difco) Blood Agar Base powder' 40. Og 

Soluble starch 20. Og 

Distilled Water 1000 mL 

Preparation: 

- Slowly , add 20g soluble starch to 1000 mL distilled water. 

- Add 40g Blood Agar Base to the mixture. Heat the solution to 90° C to 
dissolve. 

- Autoclave 15 min. , 121° C. 

- Pour in a laminar flow hood into round petri dishes 100 mm x 15 mm. 

- Refrigerate plates. 

The fecal streptococci isolates were tested for starch hydrolysis by placing a 
1 line streak on a 6 sector plate. The plates were incubated at 37° C for 5 
days. At which time they were flooded with Grain's Iodine solution and checked 
after 30 min. A clearing of the media indicates a positive starch resiilt. 

Ifedium - Todd-Hewitt Broth 

Ingredients: 

(Difco) Todd Hewitt Broth Powder 30. Og 

Distilled Water 1000 mL 

Preparation: 

- Mix powder into distilled water 

- Dispense into tubes (5 ml/tube) 

- Autoclave, 15 min., 15 psi (121° C) . 

Inoculation: 

- For testing growth at 10° C and 45° C using a needle, transfer seme 
growth from the plate into the broth - No visible turbidity should be 
detected after inoculation. 

Incubation: 

- 10° C up to 5 days 

- 45° C 



- 329 - 

Reading: 

- 10° C and 45° C 

- Positive, - visible growth in broth 

- Negative, - clear non turbid broth 

Madium - TFY 

Ingredients: 

BBL Trypticase 10. Og 

BBL Fhytone 5.0g 

Glucose 5.0g 

Difco Yeast Extract 2.5g 

Tween 80 1 aiL 

Cysteine hydrochloride 0.5g 

K2HPO4 2 . Og 

^^2-6H20 " 0,5g 

ZnS04 . 7H2O , . 25g 

CaCl2 0.15g 

FeCl3 trace 

Difco Bacto, i^gar 15. Og 

Distilled Water 1000 mL 

Preparation: 

- Final pH is 6.5 after autoclaving at 121° C for 25 min. 

- The media was poured under a lamincur flow hood into round petri dishes 
100 mm X 15 inm. 



This media was used to isolate, cultivate Bifidobacteria either by spread 
plating or membrane filtration. 

Plates were incubated anaerobically in an anaerobic jar using BBL Gas Pack 
anaerobic systems (70304) with BBL Gas Pack indicator strips (70504) for 43 hr. 
at 37° C. 

Medium - Urease Reagent 

Ingredients: 

Urea 10 . Og 

Phenol Red Solution 0.5 mL 

l.Og of phenol red in 10 mL ethanol (0.25 in 2.5 mL) 
Distilled Water 500 mL 

Preparation: 

- Weigh the phenol red in a weigh boat. Add the alcchol and ni:< zc 
dissolve the dye. 

- Weigh the urea and add it to the distilled water. 

- ?dd 0.5 mL of the prepared phenol red solution to t.^.e 'area soluricn. 

- Adjust pH to 5.0 + 0.2 



- 330 - 

- Store in a pyrex container for up to 2 weeks in the refrigerator. 

- Check (and adjust) pH before use. 

The urease test is perfomved to determine the presence of E. coli . 

After the m-Tec (media used to enumerate Tecal coliforms) is counted, the 
filter is placed on a pad, contained in a small petri dish, soaked with urease 
reagent (but not dripping) . The filter is left for 15 min. and recounted. 
Yellow-yellow green colonies are indicative of E. coli . 

Medium - YN17(b) (MF media for Bifidobacteria) 

Ingredients: 

Yeast extract 20. Og 

Polypeptone 10 . Og 

Lactose • 10 . Og 

Casamino acid 8.0g 

NaCl 3 . 2g 

Bromocresol green 0.30g 

Cysteine hydrochloride 0.40g 

(Difco) Bacto-agar 15. Og 

Distilled Water 1000 mL 

Preparation: 

- Stir on medium heat until agar dissolves. 

- Autoclave for 15 min. at 121° C. 

- Adjust pH to 6.9 + 0.1. 

- Cool to 60° C. and add: 

Ncilidixic acid 0.03g 

Kanamycin sulphate O.OSg 

Polymixin B sulphate 0.0062g 

Target colonies on YN17 (b) appear dark blue and black. 

*YN17 growth media 

Follow above recipe. However, do not include bronocresol green or antibiotics.